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dissertation entitled

CHARACTERIZATION OF FOUR MEMBERS OF THE
FERROUS ION AND a-KETOGLUTARATE DEPENDENT

HYDROXYLASE FAMILY FROM TRYPANOSOMA BRUCE]:

TWO THYMINE HYDROXYLASE-LIKE PROTEINS, J-
BINDING PROTEIN 1, AND AN ALKB HOMOLOG

presented by

JANA M SIMMONS

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Biochemistry and Molecular
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CHARACTERIZATION OF FOUR MEMBERS OF THE FERROUS ION AND a-
KETOGLUTARATE DEPENDENT HYDROXYLASE FAMILY FROM
TRYPANOSOMA BRUCE]: TWO THYMINE HYDROXYLASE-LIKE PROTEINS, J—
BINDING PROTEIN 1, AND AN ALKB HOMOLOG
By

Jana M Simmons

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Biochemistry and Molecular Biology

2010

ABSTRACT
CHARACTERIZATION OF FOUR MEMBERS OF THE FERROUS ION AND a-
KETOGLUTARATE DEPENDENT HYDROXYLASE FAMILY FROM
TRYPANOSOMA BRUCE]: TWO THYMINE HYDROXYLASE—LIKE PROTEINS, J-
BINDING PROTEIN 1, AND AN ALKB HOMOLOG
By
Jana M Simmons
Trypanosomes are eukaryotic parasites and the causative agents of several
mammalian diseases. They are phylogenetically divergent making study of their biology
pivotal to understanding their relationships to both ‘lower’ and ‘higher’ organisms.
Trypanosomes contain many open reading fi'ames predicted to encode proteins of the
Fen/(x-ketoglutarate (aKG) hydroxylase family including two thymine 7-hydroxylase-like
proteins (TLPS & TLP7), J-binding protein 1 (JBPI), and an AlkB homolog (TbABH).
TLPS & TLP7 transcript levels are 4 and 2.5 fold higher in the bloodstream form

over the procyclic form parasites, indicating a role for the respective proteins in the
former life stage. Protein production was verified in both life stages by western blot with
a polyclonal anti-TLPS antibody and suggested that these proteins may undergo some
posttranslational modification in vivo. Recombinant TLPS & TLP7 were confirmed to be
members of the Fen/qKG hydroxylase family based upon formation of a characteristic
metal—to—ligand charge-transfer (MLCT) chromophore when Feu and 0tKG were added to
anaerobic samples. A homology model of TLPS was created and a library of small
molecules docked with this model, indicating a propensity of the protein to bind

heterocyclic molecules resembling nucleotides. Unfortunately, no DNA binding or

enzymatic activity was observed for these proteins, leaving their cellular function

unknown.

JBPI is known to be involved in the hypermodification of thymine in nuclear

DNA resulting in the formation of B-D-glucosylhydroxymethyldeoxyuracil, or base I.

The first step of base I synthesis involves hydroxylation of the C5 methyl group of
thymidine, a reaction type known to be catalyzed by the Fen/OKG hydroxylases in other
organisms. Recombinant JBPI was confirmed to be a member of this enzyme family
based on the characteristic MLCT chromophore formation. Unfortunately, no enzymatic
turnover was observed by the in vitro methods employed leaving the direct detection of
activity elusive.

TbABH is a homolog of AlkB from Escherichia coli, an enzyme that oxidatively
repairs alkylated DNA, consistent with such a mechanism of DNA repair existing in
trypanosomatids. Sequence analysis suggested that TbABH is a nuclear, DNA-binding
protein, and electrophoretic mobility shift assays using recombinant TbABH
demonstrated preferential binding to alkylated double-stranded DNA. Membership in the
Fen/0K6 hydroxylase family was verified by MLCT chromophore formation, however,
attempts to detect in vi tro activity were unsuccessful. TbABH was shown to be capable
of complementing an alkB mutant of E. coli subjected to alkylation stress. The

complemented cell line was approximately 2-fold more resistant to alkylation-induced

growth inhibition than the mutant, confirming the assignment of TbABH as a functional

AlkB-like DNA repair protein in T. brucei.

To my husband and children,
with respect, gratitude, and love

iv

ACKNOWLEDGEMENTS

I am grateful to the many people who have been a part of my life and a part of my
graduate journey in the last six years. First, my utmost thanks go to my advisor, Dr
Robert Hausinger, for his ever-present and immensely helpful guidance, encouragement,
inspiration, and support. I also want to thank the members of my committee: Dr. Michael
Feig, Dr. Charlie Hoogstraten, Dr. Donna Koslowsky, and Dr. Leslie Kuhn. Special

thanks goes to Dr. Koslowsky for her assistance with several of the studies presented in
this thesis including the epitope tagging of TLPS, the DNA binding studies with TbABH,
and the qRT-PCR. I would also like to thank Dr. Kuhn for her help in optimizing and
validating the homology model of TLPS.

I have had the pleasure and privilege to work with many wonderful people in the
lab over the years including Dr. Scott Mulrooney, Dr. Tina Miiller, Dr. Piotr Gryzska, Dr.
Lee Macomber, Dr. William Kittleman, Dr. Hajime Masukawa, Dr. Soledad Quiroz, Dr.
Meng Li, Dr. Efthalia Kalliri, Dr. Jong Kyong Kim, Mukta Rohankhedkar, Eric Carter,
Jodi Boer, Nicholas Flugga, Saundra Hempel, Greg Moyerbrailean, Mike Howard,
Megan Andrzejak, Brittnie DeVries, Scott Taber, Chris Towns, Rachel Morr, Andrea
Silva, Bruce Fraser, Kim Anderson, Melody Campbell, Scott Elsey, and Matt Fountain.
For all of their camaraderie, technical help, and support, I am grateful. I want to
especially thank Piotr for all his help with the spectroscopy studies, Greg for his help

maintaining the parasite cultures, William for his help with the mass spectrometry
studies, Megan for help with the EMSAs using abasic site-containing DNA, and Mike for

performing the docking studies with TLPS.

I also need to thank Dr. Henri van Luenen of the Borst lab in the Netherlands for
providing JBPI protein and collaborating on those studies. I would like to thank Dr. Joe

Beckmann for encouraging and motivating me and giving me excellent training at Alma

College before I began my Ph.D. studies.

I cannot adequately express my love and gratitude toward my family for their
support and encouragement. To my husband, Aaron, I could have never done this
without you. Finally, I thank God for granting me the ability to undertake such a

challenging journey, and the fortitude to complete it.

vi

TABLE OF CONTENTS

LIST OF FIGURES ...................................................................................... - ............. i x
ABBREVIATIONS ................................................................................................... xi
CHAPTER 1
Introduction ................................................................................................................ l

1.1 Overview of trypanosome biology .................................................................. 2
1.1.] T brucei life cycle .................................................................................. 3
1.1.2 Antigenic variation .................................................................................. 5
1.1.3 General features of kinetoplastids ........................................................... 5
1.1.4 T. brucei replication, chromosome arrangements, and transcription ...... 6
1.1.5 Subtelomeric expression of VSG genes .................................................. 11
1.2 F e"/aKG-dependent hydroxylases .................................................................. 14
1.2.1 Mechanism of Fe I/oiKG hydroxylases .................................................... 14
1.2.2 Hydroxylases in nucleic acid and chromatin metabolism ....................... 18
1.3 Organization of thesis and open questions ...................................................... 20
References .................................................................................................................. 22
CHAPTER 2
Trypanosoma brucei brucei: Thymine 7-hydroxylase-like proteins ......................... 28
Abstract ...................................................................................................................... 29
2.1 Introduction ..................................................................................................... 30
2.2 Materials and Methods .................................................................................... 32
2.2.1 Candidate gene identification ................................................................. 32
2.2.2 Parasites and quantitative real-time PCR ................................................ 32
2.2.3 Construction of TLPS- and TLP7—expression Escherichia coli strains ..33
2.2.4 Over-expression and purification of TLP5 and TLP7 ............................ 34
2.2.5 Antibody production and Western blotting ............................................. 35
2.2.6 Spectroscopy ........................................................................................... 36
2.2.7 Enzyme assays ........................................................................................ 36
2.3 Results and Discussion ................................................................................... 38
References .................................................................................................................. 47
CHAPTER 2 APPENDIX
2A.1 Introduction .................................................................................................. 51
2A.2 Oligomeric state determination .................................................................... 52 .
2A.3 Homology model .......................................................................................... 54
2A.4 Relocation of purification tag ...................................................................... 58
2A.S C-terminal truncation ................................................................................... 59
2A.6 DNA binding ................................................................................................ 60
2A.7 In vivo epitope tagging of the TLP5 gene .................................................... 61
2A.8 Pull-down assays with TLP5 and trypanosome cell extracts ....................... 66

vii

2A.9 Conclusions .................................................................................................. 67

References .................................................................................................................. 68
CHAPTER 3
Search for hydroxylase activity in the trypanosomal protein, JBPI .......................... 69
3.1 Introduction ..................................................................................................... 70
3.2 Materials and Methods .................................................................................... 74
3.2.1 Determination of succinate production as a measure of uncoupled
enzymatic turnover .................................................................................. 74

3.2.2 Oxygen consumption as a measure of uncoupled enzymatic tumover...76
3.2.3 Spectroscopic detection of a metal-to-ligand charge-transfer

feature ..................................................................................................... 76
3.3 Results and Discussion ................................................................................... 78
References .................................................................................................................. 81
CHAPTER 4
Characterization of a T typanosoma brucei AlkB homolog capable of repairing
alkylated DNA ........................................................................................................... 83
Abstract ...................................................................................................................... 84
4.1 Introduction ..................................................................................................... 85
4.2 Materials and Methods .................................................................................... 91
4.2.1 Gene identification and multiple sequence alignment ............................ 91
4.2.2 Cloning .................................................................................................... 91
4.2.3 Protein production and purification ........................................................ 92
4.2.4 Gel filtration chromatography ................................................................. 93
4.2.5 Spectroscopy ........................................................................................... 93
4. 2. 6 Electrophoretic mobility shift assays ...................................................... 94
4. 2. 7 In vitro enzyme assays ............................................................................ 97
4.2.8 Complementation assays ......................................................................... 98
4.3 Results and Discussion ................................................................................... 101
4.3.] Analysis of the TbABH sequence ............................................................ 101
4.3.2 General biochemical properties of TbABH ............................................ 103
4.3.3 DNA binding by TbABH ........................................................................ 105
4.3.4 Examination of DNA repair by TbABH ................................................. 108
4.3.5 Conclusions ............................................................................................. 112
References .................................................................................................................. 113
CHAPTER 5
Perspectives ................................................................................................................ 1 18
5.1 Introduction ..................................................................................................... 119
5.2 Summary of Results ........................................................................................ 120
5.2.1 TLP5 and TLP7 are unlikely to participate in pyrimidine salvage ......... 120
5.2.2 JBPl is inactive in its recombinant form ................................................ 121
5.2.3 TbABH is a DNA-binding protein homologous to E. coli AlkB ............ 122
5.3 Conclusions and Future Directions ................................................................. 124
References .................................................................................................................. 127

viii

LIST OF FIGURES

(Images in this dissertation are presented in color)

CHAPTER 1
1.1 Anatomy of T Iypanosoma brucez' .................................................................. 8
1.2 Schematic of discontinuous transcription and the production of mature
mRNA by trans splicing ................................................................................. 10
1.3 Bloodstream form subtelomeric expression site organization ........................ 13
1.4 General mechanism of F e" and aKG dependent hydroxylases ....................... 16
1.5 Reaction schemes for nucleic acid modifying hydroxylases .......................... 19
CHAPTER 2
2.] Sequence comparison of TLP5, TLP7, and T7H from R. glutinis .................. 39
2.2 Quantitative real-time PCR analysis of TLP5 and TLP7 expression in BF
and PF stages of T. brucez' ............................................................................... 40
2.3 Electrophoretic analysis of purified TLP5, TLP7, and extracts of PF and
BF cells ........................................................................................................... 43
2.4 Spectroscopic evidence for binding of Fe" and aKG by TLP5 ...................... 45
CHAPTER 2 APPENDIX
2A.1 Native size and Oligomeric state determination of TLP5 ............................. 53
2A.2 Homology model of TLP5 ........................................................................... 56
2A.3 Results of docking a subset of the ZINC small molecule database into the
modeled active site of TLP5 ........................................................................ 57
2A4 Hemagglutinin epitope tagging of TLP5 ..................................................... 65
CHAPTER 3 '
3.1 Synthesis of base I .......................................................................................... 72
3.2 uKG and succinate detection by HPLC .......................................................... 75

ix

3.3 Spectroscopic evidence for binding of F e" and aKG by JBPl ....................... 80

CHAPTER 4

4.1 Reaction ofAlkB with lmeA and 3meC in DNA or RNA ............................. 87

4.2 Structure of AlkB (PDB 2fd8) ........................................................................ 88

4.3 Multiple sequence alignment of selected bacterial and eukaryotic AlkB-like
proteins ............................................................................................................ 102

4.4 Native size and Oligomeric state determination of TbABH ............................ 104

4.5 Spectroscopic evidence for binding of Fe" and aKG by TbABH .................. 106

4.6 EMSA studies of TbABH and various DNA substrates ................................. 107

4.7 Time course viability test with wild-type and AalkB BW251 13 cell lines
stressed with 0.5% MMS ................................................................................ 110

4.8 Complementation of an E. coli alkB mutant with TbABH under alkylation
stress ................................................................................................................ 111

1 meA
3meC
aKG
ABHl
ABH2
ABH3
ApoLl
ATP

Base J

BC IP/N BT

BF

CF U
Cth3
dNTP
ds
EDTA
EMSA

ESAG

GC/MS

HA

HPLC

ABBREVIATIONS

l —methy1-adenine

3-methyl-cytocine

alpha-ketoglutarate

AlkB homolog 1

AlkB homolog 2

AlkB homolog 3
apolipoprotein Ll
adenosine triphosphate
[3-D-glucosylhydroxyrnethyldeoxyuraciI
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chloride
bloodstream form
colony forming unit
cytochrome c3
deoxy-nucleotide triphosphate
double stranded
ethylenediamine tetraacetic acid
electrophoretic mobility shift assay
expression site associated gene

gas chromatography/mass spectrometry
hemagglutinin

high performance liquid chromatography

xi

HOMedU

IPTG

JBPl
JPBZ
ijC

kDa

LB
MLCT
AMMS
NAD*an
NCBI
NLNTA
OPDA
ORF
PCR

PDB

PF

PVDF
RNAP
RNAi

RT

SDS-PAGE

hydroxymethyldeoxyuracil
isopropyl thiogalactopyranoside
see base I

J-binding protein 1

JBPl related protein

Jumonji C

kilodalton

lysogeny broth

metal-to-ligand charge-transfer
methylmethane sulfonate
nicotine adenine dinucleotide (oxidized or reduced)
National Center for Biotechnology Information
nickel-bound nitrilotriacetic acid
ortho-phenylenediamine

open reading frame

polymerase chain reaction
protein data bank

procyclic form

polyvinylidene fluoride

RNA polymerase

RNA interference

reverse transcribed

sodium dodecyl sulfate - polyacrylamide gel electrophoresis

xii

SL RNA splice leader RNA

33 single stranded

SWI-2/SNF-2 switch/sucrose nonfermentable

TauD taurine/aKG dioxygenase

TbABH T rypanosoma brucei AlkB homolog

T7H thymine 7-hydroxylase

TLP5 thymine 7-hydroxylase-like protein (from chromosome 5)

TLP5AC13 13 residue C-terminal truncation of TLP5
TLP7 thymine 7-hydroxylase-like protein (from chromosome 7)
UTR untranslated region

VSG variable surface glycoprotein

xiii

CHAPTER 1

INTRODUCTION

This dissertation focuses on trypanosomal enzymes of the non-heme iron (Fen)-
and a—ketoglutarate (aKG)—dependent hydroxylase family, with particular emphasis on
proteins that act on nucleic acids and those catalyzing the modification or repair of DNA.
To provide a framework for understanding these studies, I divide my introduction into

two major sections; first I discuss trypanosomes and their unique biology including key
features of gene expression, and then I describe the Fell/(1K6 hydroxylase family

emphasizing its members involved in nucleic acid modification.

1.1. Overview of T rypanosome Biology
Trypanosomes are eukaryotic unicellular parasites that are the causative agents of

several diseases affecting mammals including Human Afiican Trypanosomiasis
(commonly called Sleeping Sickness), Chagas disease in humans, and Nagana in cattle
(1). These diseases affect millions of people each year and pose a heavy economic
burden to the endemic countries due to the loss of livestock. The World Health
Organization estimates near 100,000 new cases of Sleeping Sickness each year with only
a fraction of those being reported or treated since the most affected populations are
mainly rural and among the poorest in Africa. Illustrative of the severity of the problem,
the BBC World News produced a documentary aired on October 11, 2008 calling
Sleeping Sickness “The Deadliest Disease” on Earth (2)
(http://www.survival.tv/documentaries/sleeping_sickness.php). Given the prevalence of
trypanosomal diseases, there is great interest in the development of drugs to combat

infection. Still, treatments are sorely limited and continued active research is needed to

better understand these organisms and to develop improved methods of control. For

example, detailed biochemical analyses of enzymes specific to trypanosomes could
suggest targets for future drug development.

Trypanosoma sp. are members of the order kinetoplastidae which also include
organisms of the genera Leishmania, responsible for additional vertebrate diseases (3),
and Crithz'dia, responsible for various invertebrate diseases (4). The Kinetoplastids are a
phylogenetically distinct group that diverged early from the central eukaryotic domain
before the branching of fungi, plants, and animals (5,6). Therefore, study of their
physiology and biological processes provide useful insight not only for the purpose of
disease treatment and prevention, but also for understanding the development of this
ancient branch of life. Because my work involves Trypanosoma brucei, the following '

sections focus on this microorganism.

1.1.1. T. brucei Life Cycle
T. brucei has a complex multi-phase life cycle including a morphologically

distinct proliferative stage in an insect vector (Tsetse fly) and another in the targeted
mammalian host. The insect stage or procyclic form (PF) of the parasite begins in the gut
of the fly when the insect takes a blood meal from an infected mammal. Most parasites
are cleared from mature insects and very few actually end up transmitting the
trypanosomes to a new host (7). Surviving parasites migrate to the salivary glands of the
fly over the course of several days where they transform into a non-proliferative

metacyclic form awaiting transfer to a new mammalian host.

When the T. brucei-carrying fly bites another mammal, the trypanosomes are

introduced into the bloodstream and another major life stage of the parasite begins. The

bloodstream form (BF) is the stage where human disease is manifest. There are several

forms of disease caused by the strains of T. brucei. T brucei brucei is the strain that
infects livestock since humans have an innate immunity to this particular parasite due to
apolipoprotein L1 (ApoLl), also known as “trypanosome lytic factor” (8). T. brucei
rhodesiense is most prevalent in eastern Africa and causes an acute form of the disease in
humans that often is fatal after only a few weeks of infection (1). T. brucei gambiense is
most prevalent in central and southern Africa and causes a chronic form of the disease
where the human victim may go undiagnosed for weeks to months (1). In a chronic
infection, the parasites eventually cross the blood-brain barrier and colonize the
cerebrospinal fluid while also invading other internal organs. Personality changes,
impaired mental functions, and severely disrupted circadian rhythms (hence the term
Sleeping Sickness) are all symptoms of this second stage trypanosomiasis. If left
untreated, the infection leads to coma and death (9).

The ability of T. b. rhodesz'ense and T. b. gambiense to effectively colonize
humans relates to a serum-resistance-associated gene (10,11). In particular, humans
produce ApoLl which is taken up into the trypanosome cell as part of a high density
lipoprotein complex with haptoglobin and hemoglobin (12,13). ApoLl associates with
the cell’s lysosome, forming pores in the membrane and leading ultimately to cell lysis
(8). T. b. rhodesz‘ense and T. b. gambiense produce the serum-resistance-associated

protein from an active variable surface glycoprotein (VSG) expression site which binds to
the C-terminus of ApoLl and neutralizes this effect. Recently, it was shown that minor
mutations to the C-terrninal helix of ApoLl can ablate this resistance in T. b. rhodesiense

making it sensitive to recombinant ApoLl in vitro and in transfected mice (14).

1.1.2. Antigenic Variation

In order to maintain an effective colonization of the host, the strictly extracellular
parasite must continually deal with the immune system responses to which it is exposed.
BF T. brucei utilizes a process called antigenic variation that allows the trypanosome to
evade the host immune system by periodically changing its expressed VSG coat
glycoprotein in a coordinated fashion (10). The genetic arrangement of the VSG—
encoding regions and process of expression will be discussed in section 1.1.5. In the PF
trypanosomes the surface coat is dominated by proteins called procyclins (or procyclic
acidic repetitive proteins). Unlike the BF protists, the PF cells can express several
different procyclins at one time and these proteins remain invariant through the course of
this life stage. There are two major forms of procyclins termed EP and GPEET. These
designations refer to the single amino acid code for the residues found in the repeated

regions of the glycoproteins (15).

1.1.3. General Features of Kinetoplastids

T. brucei’s surface coatings in its two life stages are characteristic of this species,
whereas other features of their biology are shared more broadly by the order
kinetoplastidae. Namely, members of this order contain a single mitochondrion (Figure
1.1.6) containing a DNA compartment termed a kinetoplast (Figure 1.1.7) that has a
unique genetic content made up of several maxicircles and several hundred concatenated
minicircle DNAs. Through elaborate mechanisms beyond the scope of this introduction,

these serve to generate the required mRN As to allow expression of mitochondrial

proteins. In T. brucei, the function of the mitochondrion varies greatly throughout the
life cycle. In PF parasites, the mitochondrion is large and active with a functional
tricarboxcylic acid cycle responsible for generation of ATP. In contrast, nutrients are
plentiful in the environment of the host bloodstream where the parasite’s mitochondrion
is greatly reduced in size and quiescent. Glycolysis alone provides ATP to the protist and
pyruvate is excreted from the cell (9).

All kinetoplastids are characterized by a single flagellum that extends down the
length of the parasite and provides for motility (Figure 1.1.2). The flagellar pocket
includes a portal providing the sole means for import and export of cellular metabolites
and proteins across the cell wall (Figure 1.1.4) (16). This exclusivity is due in part to the
previously mentioned dense coating of glycoproteins covering the rest of the cell surface.
In addition to the dense protein coat precluding access from the outside, the subpellicular
microtubules are too closely spaced to allow transport vesicles access to the plasma
membrane from the inside (Figure 1.1.3). T. brucei undergoes very high rates of non-
specific, exclusively clathrin-dependent endocytosis through this pocket so that the
surface proteins are constantly recycled. BF parasites also express several transferrin
receptors in the flagellar pocket which are responsible for uptake of host ferritin and

provide the iron needed by the cell (16).

1.1.4. T. brucei Replication, Chromosome Arrangements, and Transcription
T. brucei replication exhibits several unique properties compared to more typical
eukaryotes. They contain single c0pies of their Golgi complex (Figure 1.1.9) and

lysosome (Figure 1.1.10), thus creating a challenge for replication as each of these

organelles needs to be copied and dutifully partitioned to the daughter cells (1 7). The
nuclear genetic arrangement in these cells is very streamlined, and the kinetoplast nearly
lacks intronic regions and possesses some overlapping genes. T. brucei contains 11
linear megabase-sized chromosomes containing the vast majority of the protein-coding
genes and gene arrays, several intermediate chromosomes that contain repetitive elements
and occasionally a VSG expression site, and up to hundreds of mini-chromosomes
containing mostly pseudogenes and repertoires of VSG-encoding genes amongst highly
repetitive sequences (18,19). The latter regions are believed to provide a repertoire of
nearly inexhaustible options for recombination to create new varieties of VSGs.
Trypanosomes are diploid for their large chromosomes. These contain most, if
not all, protein-encoding genes except for some VSG-encoding genes. The exact sizes of
the chromosomes vary greatly between trypanosome populations due to large repetitive
elements and tandem gene arrays, which give rise to high rates of recombination and
unequal crossing over events. Mini-chromosomes also vary greatly in size as they are
made up of typical eukaryotic telomeres linked by large tracts of a 177-bp repeat with
usually one VSG-encoding gene or pseudogene. The ploidy of the mini-chromosomes is

less certain due to a lack of distinct chromosomal markers (18).

Figure 1.1: Anatomy of T rypanosoma brucei. l. nucleus 2. flagellum 3. subpelicular
microtubules 4. flagellar pocket 5. endoplasmic reticulum 6. mitochondrion 7. kinetoplast
8. basal body 9. Golgi body 10. lysosome. Figure adapted with permission from Dr.

Markus Engstler.

 

One thing markedly lacking from the nuclear genome of T. brucei and other
trypanosomatids is the presence of typical RNA polymerase (RNAP) II promoters.
Conventional eukaryotic protein-coding gene transcription involves the formation of
initiation complexes that congregate at an RNAP 11 promoter with many genetic
regulatory elements affecting the transcription initiation of each open reading frame
(ORF) (20). In contrast, trypanosomes have long poly—cistronic genetic arrangements on
each chromosome with a strand switch region somewhere in the middle from which
transcription initiates and radiates outwards toward the telomeres, with one set of ORFs
on one strand and another set on the other (21). This leaves only the 5’ and 3’
untranslated regions between the ORFs to provide regulation, almost all of which occurs
post—transcriptionally (22). Therefore, gene products needed in abundance, such as
tubulin, are arranged in repetitive gene arrays in order to obtain greater amounts of the
mRNA. That mRNA is then stabilized to allow for greater levels of translation. Due to
this unique mechanism for generation of transcripts, trypanosomes process pre-mRNAs
by trans-splicing. As depicted in Figure 1.2, a 39—nucleotide spliced leader sequence
(SL-RNA) is transcribed from the RNAP II promoter (using an RNAP that is altered in
its subunit composition when compared to that involved in poly-cistron transcription) in
great abundance (100-200 tandem copies per genome) and is trans-spliced onto the 5’
end of every mRN A produced in the nucleus. These are then polyadenylated based on

the location in the intergenic region of the 5’ splice acceptor site (23).

Figure 1.2 Schematic of discontinuous transcription and the production of mature mRNA
by trans splicing. Transcription of short discrete tandemly repeated genes (SL RNA) and
polycistronic protein-coding regions (exemplified by L. major chromosome 1 (24)) by
RNAP H is indicated at the left. The primary transcripts from the two classes of gene are
shown in the center intersecting with steps in the trans splicing pathway (right panel).
The bimolecular trans splicing reaction resembles the unimolecular cis splicing pathway
in many aspects, including the initial cleavage of the SL RNA splice donor site, the
formation of 5’—2' phosphodiester bond on a branch—point A residue, cleavage at the pre-
mRNA splice acceptor site and ligation of the two exons. Differences between the two
processes include the formation of a Y-branched intermediate (rather that a lariat-like

intermediate) and the coupled polyadenylation of the upstream mRNA. Figure adapted

 

with permission from (23).

SPLICED LEADER
GENE ARRAY , SLf RNA

 

 

   

Q) _

 

 

 

m7G[-—m7rl

 

 

   

 

 

 

  

 

 

   

monomsrnomc
._~ - TRANS-SPLICING
TRANSCRIPTION ‘5'
AT Two Exrraames
PoLYCISTReuie' G_AM m7cl

‘ » H POLYADENYLATIO

LEISHMANIA FRIEDLIN P°'-Y°'5TR°N TRANSLATION
CHROMOSOME 1 or GENE *x'

mRNA

v

10

1.1.5. Subtelomeric Expression of VSG genes
Another unique genetic feature of T. brucei relates to its expression of genes in

subtelomeric regions of the megabase chromosomes. These regions, spanning up to 2 kb
upstream of the telomeric repeats, include the VSG expression sites (Fig. 1.3).
Trypanosomes contain approximately 20 of these sites which are made up of various
repetitive DNA satellites, the VSG gene, and several other expression site-associated
genes (ESAGs). These genes encode variations of transferrin receptors (ESAG 6 and 7)
for iron sequestration from the host, a homolog of a biopterin transporter (ESAG 10),
several proteins likely involved in the differentiation between life stages (ESAG 4 and 8),
other protein-encoding genes of unknown function, and the serum-resistance—associated
protein responsible for the ability of T. b. rhodesiense and T. b. gambiense to effectively
colonize humans (10,1 1,25).

During trypanosome infection, only one VSG expression site is active at any
given time, allowing the population of trypanosomes to express a single coat protein.
Transcription of the VSG gene is a—amanitin insensitive (26), implicating the
involvement of RNAP I which does not typically transcribe protein-encoding genes.
RNAP I was shown to be directly responsible for transcription of the VSG and procyclin
surface protein-encoding genes of trypanosomes (27). Although each VSG expression

site has its own RNAP I promoter and seems to initiate transcription constitutively
(28,29), transcription aborts before reaching the VSG gene in silent expression sites (30).
The silencing mechanisms employed by these parasites to accomplish such

coordinated gene expression have been studied for many years, though they remain

ll

largely a mystery. Positioning effects mediated by the subtelomeric localization have
been investigated with some contradictory results, but it does seem that there is some
promoter restraint, especially in BF trypanosomes (31,32). In addition, a modified base
found in telomeric and subtelomeric regions has also been implicated in gene silencing.
B-D—glucosylhydroxymethyluridine, known as base I (33), replaces up to 1% of the
thymidine in trypanosomal DNA (34). This novel base is localized to telomeric repeats,
subtelomeric repeats surrounding the VSG expression sites, and sequences within VSG
expression sites not being expressed (Fig. 1.3). However, the one active VSG expression
site is void of the modification (35). Further, the active VSG expression site is
sequestered in the “expression site body”, an area of the nucleus that is separate from the
nucleolus; this subnuclear localization may also play a role in its discriminatory
expression (36).

Trypanosomes can switch the expression of their VSG by several mechanisms.
The most common mode of switching is by keeping the same active VSG expression site
and swapping out the VSG gene with another by homologous recombination.
Alternatively, trypanosomes can splice together new VSGs by recombining part of the
currently expressed VSG with a pseudo gene from a minichromosome or intermediate
chromosome. Another mechanism is to silence the current VSG expression site while
activating another. This approach may be less common as it would require more
chromatin rearrangement, though it would also allow the expression of variant transferrin

receptors and other ESAGs.

12

Figure 1.3 Bloodstream form subtelomeric expression site organization Figure adapted
from (25). ESAGs are depicted by numbered open boxes (those genes present in only
some expression sites are shown in parentheses). Relative location of base I is shown
above the expression site diagrams and is based on immunoprecipitation of fragmented
DNA with an anti-J antibody (3 5). Filled triangles represent 50 bp repeats, hatched boxes
represent 70 bp repeats, and filled ovals represent telomeric repeats. The top diagram
depicts a silent expression site with base J found throughout the coding sequences while
the center diagram depicts an active expression site, devoid of base I and fully .
transcribed (dashed arrow). Finally, the bottom diagram depicts a specialized expression

site of T. b. rhodesiense containing the serum resistance associated gene.

   

 

 

 

 

J J I J JJJ JJJJJJJJJJJJ
8 3 2 11 a uummm-

J JJ _____,__-_____ ___________________ JJJJJJJ
a 3 11 1 9 vse

 

 

 

 

 

 

 

 

 

 

 

 

 

 

J JJ JJJJJJJJJJ
l. B 5 111111111 Mlllllllll

 

l3

1.2. F ell/aK G-Dependent Hydroxylases.

The F eu/aKG-dependent hydroxylases use a mononuclear ferrous ion to catalyze
the oxidative decarboxylation of aKG while activating molecular oxygen in order to
hydroxylate a target substrate. This broad enzyme superfamily has representatives
throughout the domains of bacteria and eukaryota, with other members found in viruses,
and they catalyze a wide variety of reactions (3 7),(3 8). These enzymes are characterized
by a double-stranded fl-helix core fold, often termed a B—jellyroll structure that is made up
of 8 [3 strands within which lies the active site (39). Section 1.2.1 will discuss the overall
reaction mechanism and Section 1.2.2 will focus on those members whose primary

function is the modification of nucleic acids or chromatin.

1.2.1. Mechanism of F e1 1/ aK G hydroxylases.

The current view of the enzyme mechanism for these enzymes, summarized in
Figure 1.4, is a modification of that first proposed over 25 years ago (40). Two histidines
and one carboxylic acid residue (usually an aspartate) coordinate one face of the iron
atom (41). This “facial triad” usually occurs in a His-X—Asp/Glu-Xn-His motif where n is
typically around 50 residues, with the remaining 3 coordination sites on the metal atom
taken up by water molecules in the resting state (Fig. 1.4 panel A). Upon addition of the
cosubstrate, uKG, two waters are displaced by the bidentate binding of the aKG molecule
via its C1 carboxylate and C2 keto groups (Fig. 1.4 panel B). The C5 carboxylate of
aKG often is stabilized by a conserved arginine located approximately a dozen residues

C-terminal to the second His residue of the facial triad (38). This enzyme state leaves

14

one iron coordination site occupied by a water molecule that is displaced by the binding
of the primary substrate (Fig. 1.4 panel C).

The two aKG-bound states can be visualized spectroscopically. Thus, in the
absence of primary substrate and oxygen, but the presence of iron and aKG, these
enzymes exhibit a metal-to-ligand charge-transfer electronic transition that is observed at
around 500 to 530 nm (42). For example, taurine/aKG dioxygenase (TauD), a well-
studied member responsible for the oxidative cleavage of taurine to release sulfite during
sulfur starvation in Escherichia coli, forms this characteristic chromophore at 530 nm
with an extinction coefficient of 140 M'1 cm'1 (42). This feature can be used to diagnose
proper protein folding and cofactor/cosubstrate binding. It is also used to confirm
membership in this enzyme family for proteins without a verified function or a known
primary substrate. Once the primary substrate binds to the protein, near the active-site
but not coordinating the iron atom, this feature often shifis. In the case of TauD, taurine
binding causes a 10 nm shift in the absorbance to higher energy and increases the
intensity of the chromophore to give an extinction coefficient of 180 M'l cm'1 at 520 nm
(42).

Displacement of the final water molecule leaves an open coordination site on the
iron atom that provides a site for molecular oxygen to coordinate the iron. This process
likely creates two very short-lived species including an F em-superoxo species and a
bridging Few-peroxo species that have never been observed (Fig. 1.4 panels D and E).
Decomposition of the latter species leads to cleavage of the cosubstrate into the products

succinate and C02 as well as the formation of a highly reactive Few-0x0 species which

15

Figure 1.4 General mechanism of Fe" and uKG dependent hydroxylases. Primary

substrate is generalized and abbreviated by R-H.

 

 

 

R+I R H
'O o B H210 ' O’ o C
ASP""-Fell/O O ASp""'Fe"\/ /
0 His’ H"\O/ H 0 His, lilrso
_ IS - 2 "
03/1 COO “A
2 H20 R‘H
A H2|O r ‘02 “D
ASP"; Fell {H20 ASP'M em /0 O
HIS i H20 HISI I \0/
HS l. His J -
z R-OH COO
K -0 + I
3 H20
R OHCOO— R-H
G OH" O 'O—-O E
ASp"--.. III Asp: iv’: 0
Fe\- Fe
His/ I OJLL His’ |\'o
HIS coo R‘H His

F 0: COO
Asp' ..... Fl l IV 0
. / e\

16

was directly observed first for TauD (43 -45), and later for prolyl hydroxylase (46) and a
related halogenase, Cth3 (47) (Fig. 1.4 panel F).

The highly reactive F e‘V oxo species then abstracts a hydrogen atom from the
primary substrate generating a likely Fem-hydroxo species and a substrate radical (Fig.
1.4 panel G). The traditional view is that the hydroxyl radical then rebounds with the
carbon radical, thus hydroxylating the substrate (which spontaneously degrades into
sulfite and arninoacetaldehyde in the case of taurine) and regenerating the ferrous ion at
the active—site. Very recent studies suggest this step is more complex, involving an F em-
oxo species and an alkoxo species (not depicted), ultimately yielding the same products
(48). The products are then released from the active-site and are replaced by three waters
to regenerate the resting state for the enzyme (Fig. 1.4 panel A).

This reaction cycle can be tightly coupled, resulting in the utilization of
stoichiometric amounts of primary substrate, aKG, and 02 for each turnover (3 8).
However, in the absence of the primary substrate or the presence of alternate substrates or
inhibitors, these enzymes can decarboxylate aKG and activate oxygen to generate
reactive oxygen species that can pose a threat to the cell. Some of this damage seems to
be avoided by “fail safe” mechanisms involving side chain self-hydroxylation (49,50) or
reaction of the activated species with a cellular reductant rather than releasing deleterious
oxygen species into the milieu of the cell (51). This “uncoupling” of oxygen activation
and substrate hydroxylation can be observed by monitoring oxygen or aKG consumption

and succinate production by various methods and has been a useful tool to aid in the

characterization of proteins of unknown function.

17

1.2.2. Hydroxylases in Nucleic Acid and Chromatin Metabolism.

Several members of the F eu/aKG hydroxylase enzyme family utilize this
powerful oxidizing reaction to modify nucleic acids. One of the best studied enzymes in
this subgroup is AlkB, an E. coli protein, that repairs alkyl lesions on DNA and RNA
(52,53). This enzyme follows the same basic mechanism described above to oxidize
methyl adducts on the N—l position of adenine (lmeA), the N-3 position of cytosine
(3meC), and selected other lesions in the context of DNA (Fig. 1.5 panel A). The
hydroxylated methyl groups are spontaneously released from the molecule as
formaldehyde, regenerating the unmodified bases.

Multiple complexes of AlkB along with its cofactor, cosubstrate, and primary
substrates have been crystallized and show that single-stranded nucleic acids are
preferred by this enzyme with the strand bent into the active site, thus flipping the
alkylated base deep into the pocket where chemistry takes place (54). Further, this
protein has a ‘nucleotide recognition lid’ that alternates between an open and closed state
upon binding of the substrate, providing more stability for the transition state and
facilitating the enclosed environment needed for the oxygenation reaction. AlkB has
been demonstrated to function in the repair of both DNA and RNA, with methylated,
ethylated, and 1,N6-etheno modified bases. The physiological relevance of reactions
involving more bulky lesions is not clear given other pathways in the cell, such as base
excision repair; however, the substrate versatility (plasticity of the active site) suggests
that AlkB may be able to serve as a back up system of repair for these lesions (55).

Another example of nucleic acid hydroxylation is found in a firngal enzyme,

thymine 7-hydroxylase, which participates in pyrimidine salvage. This enzyme catalyzes

18

Figure 1.5 Reaction schemes for nucleic acid modifying hydroxylases. A. Predominant
reactions catalyzed by E. coli AlkB. B. Reactions catalyzed by thymine 7-hydroxylase.

HMU is 5-hydroxymethyluracil. EU is S-formyluracil.

 

 

A
NH2 NH2 NH?
”R Fe(11) d“ d“ .
1-meA AlkB * + Adenine
NH2 / NH2 NH,
+ CH “KG CO + CHZOH
FEN/ 3 O2 Succmate l \N/ HCHOI \N
1k /k N/K
til 0 til 0 I 0
dB dR dFI
3-meC Cytosine
B.
O 0H 0
I NH ' NH
N/Ko o co N/KO
H al2<G sucéinate H
thymine 02 HMU
aKG
co2
succinate
H20
0 O
HOOC OHC
l ”“ .7: l L
N/KO co2 02 ti 0
H succinate aKG
uracil-carboxylic acid FU

19

the 3-step oxidation of the free base, thymine, to uracil-carboxylic acid, through
hydroxymethyluracil and 5—formyluracil as illustrated in Figure 1.5 panel B (56). The
kinetics of the later two reactions are very slow, calling into question their physiological
relevance (57). Further studies show that the three reactions are not processive, but rather
the product leaves the active site and must rebind along with another molecule of aKG
and 02 to allow a new reaction cycle to commence (57).

Other members of this enzyme family that participate in nucleic acid
hydroxylation include xanthine hydroxylase (58,59), which converts the free base
xanthine to uric acid, and deoxyribonucleoside-Z’-hydroxylase, which converts thymidine
or deoxyuridine to the free base (60). In addition, the chromatin modifying ijC
domain-containing histone demethylases remove methyl groups from lysines in histones
(61). The aforementioned enzymes encompass a substrate range from free bases to
chromatin and highlight the versatility and utility of a well controlled highly reactive

oxygenation system in the cell for maintaining and modifying nucleic acids and proteins.

1.3. Organization of this Thesis and Open Questions

Chapter 2 of this thesis explores two thymine 7-hydroxylase-like proteins (TLP5
and TLP7) in T. b. brucei including sequence analysis, cloning, and expression of their
genes, protein purification and characterization, and the in vitro search for their activity.
Chapter 3 describes my part in the characterization of J-binding protein 1 (a thymidine
hydroxylase) of various kinetoplastids, again involving protein characterization and an in
vitro search for enzymatic activity. Chapter 4 includes the characterization of a T. b.

brucei homolog of AlkB including sequence analysis, cloning, and expression of its gene,

20

purification of the protein, and both in vitro and in vivo searches for activity. Finally,
Chapter 5 summarizes my overall conclusions and describes the remaining open

questions in the area of trypanosomal F ell/OtKG hydroxylases.

21

10.

ll.

12.

13.

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27

CHAPTER 2

TRYPANOSOMA BRUCEI BRUCE]: THYMINE 7-HYDROXYLASE-LIKE

PROTEINS

These studies were published in:

Simmons, JM, Koslowsky,

DJ, Hausinger, RP. 200
Thymine 7-Hydroxylase-

9. T Iypanosoma brucei brucei:
Like Proteins. Experimen

tal Parasitology 124: 453-458.

The quantitative reverse-transcriptase PCR experiments were performed by Dr. Donna

Koslowsky and the data analysis for the anaerobic chromophore experiment was
performed by Dr. Piotr Grzyska. Mass Spectrometry experiments were performed with
the aid of Dr. William Kettleman. Additional studies not included in the publication are

provided in an appendix.

28

 

 

ABSTRACT

Two genes from T rypanasoma brucei brucei are predicted to encode Fe”- and or-
ketoglutarate-dependent enzymes related to fungal thymine 7-hydroxylase. Transcript
levels of the thymine hydroxylase-like genes are higher in the bloodstream-forth of the
parasite over the insect form, whereas Western blot analysis indicates more cross-reactive
protein in the latter life stage. The genes were cloned, the proteins purified from
Escherichia coli, and both proteins were shown to bind F ell and a-ketoglutarate,
confirming proper folding. The isolated proteins were incubated with Fe" and a—
ketoglutarate plus thymine, thymidine, and other putative substrates, but no activity was
detected. Furthermore, no thymine 7-hydroxylase activity was detected in extracts of
procyclic or bloodstream form cells. Although the functions of these proteins remain

unknown, we conclude they are unlikely to be involved in thymine salvage.

29

2.1 INTRODUCTION

microorganism.

This work describes two T. b. brucei genes, located on chromosomes 5 and 7
(Tb927.5.300 and Tb927.7.7500, respectively), which encode full-length orthologues of
thymine 7-hydroxylase (T7H) othodotarula glutinis (3,4) and are termed TLP5 and
TLP7 for thymine hydroxylase-like proteins. T7H is a non-heme iron (F e")- and a-
ketoglutarate (aKG)-dependent hydroxylase that catalyzes sequential oxidations of
thymine (Fig. 1.58) (4,5) as part of a pyrimidine salvage pathway in some fungi (3,6).
Hydroxylation of the unactivated C7 methyl group of thymine is driven by the oxidative
decarboxylation of aKG, forming succinate and C02. Significantly, trypanosomes have
not been reported to contain a pyrimidine salvage pathway and show no mediated uptake
of thymine or thymidine (7), nor does the kinetoplastid genome contain an obvious
candidate gene encoding uracil-S-carboxylate decarboxylase, the enzyme acting
immediately downstream in the salvage pathway. Further, trypanosomes seem to have no

direct need for pyrimidine salvage as they are known to be able to synthesize pyrimidine

nucleotides (8).

30

 

 

To date, three gene products in trypanosomes are predicted to belong to the
Fell/aKG dependent hydroxylase family. One gene is suggested to encode AlkB,
involved in oxidative repair of DNA damaged by alkylation (9). Two others, encoding
JBPl and JBP2 are proposed to catalyze the hydroxylation of selected thymidine bases in
trypanosomal DNA as the first step in formation of base J, B-D-glucosyl-hydroxymethyl-
deoxyuracil (10,11). Significantly, no direct evidence for hydroxylase activity has been
reported for any of these proteins. Here, we examine whether the T LP5 and TLP7 genes

encode T7H isozymes or have alternative functions.

31

2.2 MATERIALS AND METHODS

2. 2. 1. Candidate gene identification

To identify candidate thymine hydroxylase genes, the Basic Local Alignment
Search Tool (12) was utilized to search the T. b. brucei genome with the gene sequence
of R. glutinis thymine 7-hydroxylase (4) as the query, resulting in the identification of
NCBI accession numbers XM__839611.1 and XM_841277. 1. In addition, the available
sequences of the kinetoplastid order were searched with the gene encoding uracil-5-

carboxylate decarboxylase (3), the next enzyme in the fungal pyrimidine salvage

pathway, as a query.

2.2.2. Parasites and quantitative real-time PCR
Transcript levels were compared using T. b. brucei strain 427 bloodstream form

(BF) paired with strain 427 (MITat-l) procyclic form (PF) and BF 427-2 (221) paired
with PF 427, 29-13. Briefly, total RNA was treated with turbo DNAse (Ambion)
according to the manufacturer’s directions and 1 ug of RNA was then reverse transcribed
(RT) in the presence of random hexamers (20 mM Tris 8.3, 50 mM KCl, 5 mM MgCl2, 2
mM mixed dNTPs, 0.25 units RNAsin and 2 units Seikagaku AMV reverse
transcriptase). The RT reaction was stopped by heating at 70°C and diluted with 125 pl
of 20 mM Tris, 1 mM ethylenediamine tetraacetic acid, pH 8.0. PCR amplification
reactions (SYBR Green, Bio-Rad) were set up in triplicate (primers: chromosome 5'
forward, 5’-GGT TGG GTA GAG TTG ATG AAC-3’; chromosome 5 reverse, 5’-TGG

AGG ATA ATG TAG CAT AGG-3’; chromosome 7 forward, 5’-CAC ACT ATC GCG

32

 

ATA TGC GGG AC-3’; chromosome 7 reverse, 5’-GCG GGG TAA TGC ACC ATG
CG-3’) using 2 ul of the diluted RT reaction. All reported data were normalized to B-
tubulin gene internal control (primers: forward, 5' GAC GAA GGA GGT TGA TGA

GCA GAT 3'; reverse, 5' TGA AGG TGA CAG CCA TCT TGA GTC 3') that was

included in each run.

2. 2. 3. Construction of T LP5- and T LP7- expressing Escherichia coli strains

A 963-bp DNA fragment containing TLP5 was amplified by PCR using genomic
T. b. brucei strain 427 DNA as a template with “forward” (5’-AGG ATA TA_C__C_A_T_
QQC TCA CGG CTC GAT T-3’) and “reverse” (5’-GAG CAT CCT CGA GCA TCT
TTG TTT TGC GAT G-3 ’) primers which introduce Ncol and XhoI restriction sites
(underlined), and T aq polymerase master mix kit (Promega) which leaves a single 3’
adenine nucleotide overhang. The PCR product was treated with a PCR clean up kit
(Qiagen, Inc.) and ligated into pGEM-T Easy (Promega). This was transformed into E.
coli DHSa (Invitrogen), isolated from several transforrnants, and sequenced (Davis
Sequencing). T LP5 was excised from the pGEM—T backbone by Neal and XhoI
restriction and ligated into pET28b (Novagen) which had been previously cut with the
same enzymes, putting the coding sequence in frame with a sequence encoding a C-
terrninal 6-histidine tag. This plasmid was transformed into the expression strain E. coli
BL21 (DE3).

Similarly, a 963—bp DNA fragment containing TLP7 was amplified from genomic

T. b. brucei DNA using “forward” (5’-GGA TAT ACC ATG GTA ATG AAT CAC ACT

TCG ATT CCA G-3’) and “reverse” (5’-GAG CAT CCT CGA GCA TAT TTG CCT

33

TGC G-3 ’) primers, ligated into pGEM-T Easy, and transformed into E. coli DHSa.
Plasmids isolated from several transformants were sequenced, one containing a minor
A186V change was excised from the pGEM-T backbone by using Neal and XhoI, and
this fragment was ligated into pET28b to yield a sequence encoding TLP7 in frame with
a C-terminal 6-histidine tag. The recombinant plasmid was transformed into E. coli

BL21 (DE3) for expression.

2. 2. 4. Over-expression and purification of TLP5 and T LP7
E. coli BL21 (DE3) cells containing the pET28b derivatives encoding TLP5—Hi36
or TLP7-Hi36 (hereafter referred to simply as TLP5 and TLP7) were grown at 30 °C in
LB medium supplemented with 100 ug/mL kanamycin while shaking at ~160 rpm to an
optical density of 0.4 to 0.6 at 600 nm. Cultures were induced to overexpress the desired
genes by addition of isopropyl-B-D-thiogalactopyranoside to 0.1 mM, and grown for an
additional 4 h, then harvested at 4 °C by centrifugation at ~8,000 g for 8 min. The cell
paste was either used immediately for protein purification or stored at -80 °C.
In a typical purification, 2.5 mL of binding buffer (30 mM imidazole, 10 mM
Tris, 150 mM NaCl, pH 7.9) was added per gram of cell paste for resuspension. The
protease inhibitor phenylmethylsulfonyl fluoride was added to 0.5 mM, cells were lysed
by sonication (Branson Sonifier, 3 pulses of l min each, 3 W output power, duty cycle
50%, with cooling on ice), and the cell lysates were ultracentrifuged at 100,000 g for l h.
Soluble cell extracts were loaded onto a Ni-bound nitrilotriacetic acid (NTA) column
(Qiagen) pre-equilibrated with binding buffer. The column was washed with binding

buffer until the baseline was reestablished, and proteins were released with elution buffer

34

(150 mM imidazole, 10 mM Tris, 150 mM NaCl, pH 7.9). Fractions containing the
purified proteins, as determined by denaturing sodium dodecyl sulfate polyacrylamide gel
(12%) electrophoresis (SDS-PAGE) (13) and Coomassie staining, were pooled and
exchanged back into binding buffer by using Amicon centrifugal filter units with a 10
kDa molecular weight cutoff. The concentrations of proteins were determined by using
their calculated molar absorptivities at 280 nm (44,390 M'1 cm'1 for TLP5 and 49,890 M“1
cm'1 for TLP7 according to the ExPasy protein parameters prediction server
(http://ca.expasy.org/tools/protparam.html). The proteins were stored at 4 °C and

discarded after two weeks or if precipitation developed.

2.2.5. Antibody production and Western blotting
Purified recombinant TLP5 was provided to Lampire Biological Laboratories for

polyclonal antibody production in a rabbit. The IgG fraction was isolated from the
product antisera by ammonium sulfate precipitation and stored at 4 °C. Trypanosomes
(107 cells in 20 uL cell wash buffer with 0.5 mM phenylmethylsulfonyl fluoride) were
disrupted in a bath sonicator for 20 min. The lysate was centrifuged at ~16,000 g for 20
min at 4 °C, and soluble cell extracts were removed to a clean tube and used immediately
or stored at -20 °C. Purified recombinant proteins or cell extracts of trypanosomes (both
PF and BF) were subjected to 12% SDS-PAGE and Western blots were prepared by

following standard protocols using Millipore ImmobilonP membrane. Protein bands

were transferred at 4 °C either using 100 V for 1 h or 14 V overnight. Blots were probed

with a 1:10,000 dilution of the rabbit anti-TLP5 antibody and a l:30,000 dilution of goat

35

 

 

 

anti-rabbit antibody conjugated to alkaline phosphatase from Sigma. Blots were

developed using the BCIP/NBT liquid substrate system from Sigma.

2. 2. 6. Spectroscopy

UV/visible spectra were obtained by using an HP 8453 spectrophotometer
(Hewlett Packard) equipped with circulating water bath and magnetic stirrer. TLP5 and
TLP7 proteins were concentrated in Amicon centrifugal filter units with 10 kDa cutoff to
250 uM in binding buffer. Stock solutions of 10 mM Fe" and 5 mM aKG were made by
adding the dry reagents to vials, subjecting them to repeated cycles of vacuum and argon,
and adding anaerobic water using a syringe. Protein was added to a 1-cm path length,
300 uL, black-walled cuvette and made anaerobic by the same method followed by
scanning with stirring at 9 °C. Data were corrected for baseline shifts arising from
cuvette repositioning to have a uniform absorbance at 900 nm by using the software

IGOR.

2.2. 7. Enzyme assays
Oxygen consumption assays were performed in a YSI Inc. model 5300 Clark-
style biological oxygen monitor coupled to a circulating water bath to maintain a
temperature of 33 °C. TLP5 or TLP7 was assayed at 1 uM in 25 mM imidazole, 100 mM
KCl, and 5 mM MgCl2 using variations of the standard conditions: 50 11M Fe", 500 uM
(1K0, 400 uM ascorbate, and 500 uM of the potential substrates.
Succinate production assays utilized 1 pM enzyme that was incubated with 10 pM

Fe", 10 uM ascorbate, and 100 uM aKG (and sometimes MgCl2) along with potential

36

substrates in 25 mM Tris HCl, pH 8.0, at either 28 °C (PF conditions) or 37 °C (BF
conditions) for 30 min. Aliquots (300 uL) were taken at various time points, quenched
with 5 uL of 6 M sulfuric acid, centrifuged at 13,800 g for 5 min, passed through a 0.45
pm spin filter, and 200 uL was analyzed by Aminex HPX-87H (BioRad) ion-exchange
chromatography (Waters, Corp.) Organic acids were eluted with 0.013 M H2804 as the
mobile phase and detected using a refractive index detector (Waters, Corp.) set at 35 °C
and a sensitivity level of 16. Standards of aKG and succinate eluted at 19 and 26 min,
respectively, and succinate was detectable to a lower limit of 1 nmol.

Purified TLP5 and TLP7 (both at 10 uM), and trypanosome whole cell protein
extracts from both the PF and BF life stages (from ~6 x 106 and ~5 x 106 cells,
respectively) were incubated with thymine (250 uM) in 1 mM HEPES, pH 7.4, at 30 °C
for l h with added aKG (500 uM) and ferrous ammonium sulfate (100 uM). Reactions
were stopped by addition of hot ethanol, incubated at 60 °C for 5 min, and dried in a
speed vac for 2h. Lyophilized pellets were resuspended in 100 uL anhydrous acetonitrile
and silylated via treatment with 100 1.1L N-methyl-N-(tert-butyldimethylsilyl)trifluoro-
acetarnide + 1% tert-butyldimethylchlorosilane (Pierce) for 30 min at 135 °C. The
solutions were analyzed by gas chromatography-mass spectrometry (GC-MS) using

electrospray ionization on a 30 m DB-SMS, I.D. 0.25 cm column for analysis as

previously described (4).

37

2.3. RESULTS AND DISCUSSION

266). The TLP5 and TLP7 sequences contain several short gaps (of 2, 7, 2, and 8 amino
acid residues) compared to the firngal sequence, but the significance of these gaps, if any,
cannot be ascertained because the structure of T7H is unknown.

Quantitative real-time PCR methods were used to examine the steady-state
expression levels of the two genes in both the BF and PF life stages of T. b. brucei. Due
to the high sequence identity of T LP5 and T LP7 (87% at the DNA level), none of the
available programs for primer selection in trypanosomes could be used. Therefore, gene
sequences were manually analyzed and primers carefirlly chosen in order to be unique to
each gene. Triplicate runs with mRNA containing TLP5 and T LP7 from T. b. brucei
strain 427 (MiTat 1) (BF and PF), 427 clone 221 (BF), and 427 cell line 29-13 (PF)
yielded similar results consistent with both genes being up-regulated in the BF parasite
over the insect-form by approximately 4 and 2.5 fold, respectively (Fig. 2.2). This
seemingly small effect is considered significant as only about 2% of genes in

trypanosomes have been found to be regulated at least 2 fold at the level of mRNA .

abundance ( 14).

38

Figure 2.1. Sequence comparison of TLP5, TLP7, and T7H from R. glutinis. Identities
are indicated by black shading. Putative metal- or aKG-binding residues are highlighted

by asterisks.

30 50 60
I I I -I --~I
QID vae--nprpnznrzxnnlu
arm ma--nprsrznrexnnsu

. EL cnvnnvvnxsrvnnarxx

TLP5
TLP7
RqT'IH

 

TLP5
TLP7
Rg'T'IH

 

TLP5 HP G ------- MDWQEPL ll:I RALAL‘ZEGI
TLP7 lP QVGG ------- ML! EWLHIzRALALIGI '

Rg‘l‘7H

TLP5
TLP7
Rg‘r'IH

TLP5
TLP7
RgT7H

TLP5
TLP7
R91"!!!

 

39

l

Figure 2.2. Quantitative real-time PCR analysis of T LP5 and T LP 7 expression in BF -
and PF-stages of T. b. brucei. The solid bars represent the fold increase of BF over PF in
427 (MITat-l) for the two genes. The hatched bars compare the fold increase of the BF
427 (221) over the PF 29-13. All data are normalized to tubulin internal controls that

were included in each run.

Abundance Ratio (BFzPF)

 

TLP5 TLP7

40

T LP5 and TLP7 were PCR amplified out of T. b. brucei 29-13 genomic DNA and
subcloned into pET28b for expression as His-tagged proteins in E. coli BL21 (DE3).
Sequencing of plasmids isolated from individual colonies revealed a number of
differences from the published T. b. brucei TREU 927 sequence, most involving amino
acid changes in non-catalytic areas of the protein. 0f potential interest were a frame shift
mutation in TLP5 and a nonsense mutation in TLP7 detected in multiple clones from
independent PCR amplifications suggesting that one allele may be non-functional in this
strain. This phenomenon may be due to an issue inherent to the trypanosomes being
studied. The 29-13 cell line used in this study was developed from strain 427 which has
been in laboratory culture for over 30 years and is now incapable of differentiating to the
BF. Loss of the ability to differentiate back into infective blood forms may reflect the
mutation of genes essential for function during this stage without conferring negative
selection in culture.

E. coli BL21 (DE3) cells containing the pET28b T LP5 and T LP7 derivatives were
used as the source of purified TLP5 and TLP7, typically yielding 5 mg of protein per
gram of wet cellular weight. The proteins were >95% homogeneous (Fig. 2.3 panel A,
lanes 2 and 3) and of the size predicted by the sequences (37.5 kDa when accounting for
the His tag), with slight differences in electrophoretic mobility observed. The proteins
behaved as monomers when examined by Blue Native gels (Invitrogen) or Superdex 75
gel filtration chromatography (data not shown). Both proteins precipitated when
exchanged into a low salt buffer after purification, but exchange back into the column

binding buffer provided enhanced stability.

41

Polyclonal anti-TLP5 antibody was used to perform Western blots of purified
recombinant TLP5 and TLP7 along with cell extracts of both PF and BF trypanosomes
(Fig. 2.3 panel B). The antibody detected both proteins, as expected from their close
similarity. A major cross-reactive species in both BF and PF electrOphoresed more slowly
than the TLP5/TLP7 doublet; this species may be an unrelated contaminant or it might
arise from post-translational modification of these proteins (leading to an apparent ~10-
kDa increase in size) in the trypanosome, but lacking in the E. coli expression system.
Future work could include efforts to identify this band and determine what, if any,
modifications are added to the protein in vivo. In contrast to the RT-PCR results that
indicate higher transcript levels in the BF cells, the immunological analysis suggests that
TLP5/7 is more abundant in PF than BF cells; these results are compatible with the
protein(s) being turned over more rapidly in the BF life stage or being translated at a
greater rate in the PF life stage. Of primary importance, these results indicate that both
proteins appear to be present in both life stages.

To confirm proper folding of TLP5 and TLP7, the purified proteins were
examined for their ability to form a characteristic chromophore seen in other members of
the Fen/aKG—dependent dioxygenases and associated with binding of the iron cofactor
and the aKG cosubstrate (15). The proteins were monitored by UV-visible spectroscopy
under anaerobic conditions, using low temperature to maintain stability, while titrating in
orKG and F e". As shown in Figure 2.4, anaerobic TLP5 generated the diagnostic metal-
to-ligand charge-transfer transition at 505 nm when both metal and cofactor were present.
The extinction coefficient for this feature was approximately 40 M'l cm", indicating that

the chromophore was incompletely formed compared with previously described

42

Figure 2.3. Electrophoretic analysis of purified TLP5, TLP7, and extracts of PF and BF
cells. Samples were subjected to SDS-PAGE and examined by A. Coomassie staining or
B. Western blot analysis with anti-TLP5 antibodies. Lanes: 1, molecular weight ladder; 2,

TLP5; 3, TLP7; 4, extracts of PF cells; 5, extracts of BF cells.

A B

 

 

97 -. ..
66 L

I
45 e. a '1; -';‘I ...e ...
.

31* -- . ——

21

14

 

 

 

 

 

 

43

intensities (15), but addition of more metal ions led to protein precipitation. TLP7 also
exhibited the diagnostic transition (data not shown), confirming correct folding of the
protein, but it was less stable than TLP5 and precipitation during the experiment
precluded estimation of the extinction coefficient.

Despite extensive efforts, no in vitro thymine hydroxylase activity was detected
for purified recombinant TLP5 or TLP7 when assayed by using an oxygen electrode to
monitor oxygen consumption, HPLC to assess succinate production, or GC-MS to
measure thymine hydroxylation. We cannot exclude the possibility that a post-
translational modification, not occurring in E. coli, is required for activity. It is also
conceivable that the His-tag hinders activity in the recombinant proteins or that, despite
formation of the Fell/aKG chromophore indicating appropriate folding of the active site
domains, the full length proteins do not fold properly. On the other hand, these results
are compatible with the proteins utilizing alternative substrates. Other members of the
Fen/OLKG dioxygenase family recognize a wide array of substrates ranging from small
molecules to proteins and DNA (16). Several additional small molecules were tested as
putative substrates including other bases, nucleosides, and amino acids, but again no
activity was detected. With regard to the possible use of DNA as a substrate,
trypanosomes are known to contain the hyper-modified base J in their nuclear DNA
(17,18), which requires hydroxylation of an unactivated methyl group of thymidine in
DNA to create hydroxymethyl-deoxyuracil (19,20) that is subsequently glucosylated.
Thus, we examined whether TLP5 or TLP7 reacted with polynucleotides or thymidine;
however no such activity was observed. Furthermore, neither protein bound to linear

DNA fragments under any conditions examined (incubation with or without 50 uM Fe",

44

Figure 2.4. Spectroscopic evidence for binding of F e" and aKG by TLP5. The
anaerobic UV/visible spectrum of TLP5 (250 uM in binding buffer) was examined for the
sample as isolated, after adding 1 mM aKG, and while titrating in FeII with stirring at 9
°C. The (F ell-aKG-protein minus aKG-protein) difference spectra shown correspond to

the addition of 0.5, 1, 2, and 3 equivalents of metal ions.

 

 

 

 

0.015 —
g A
It:
.9
h-
0
1n
.n
< 0.005 A
<
o . r
350 450 550 650 - 750

Wavelength (nm)

45

150 uM aKG, and 100 uM MgCl2 at 37 °C for 30 min) according to gel band shift
studies. Thus, TLP5 and TLP7 are unlikely to possess DNA modifying activity,
consistent with the recent proposals that J -binding protein 1 (JBPl ), identified on the
basis of its ability to bind to J -containing chromatin, and JBP2, related to JBPI, are
critically involved in base J synthesis (10,21-24).

Extracts of PF and BF cells were directly assayed for thymine hydroxylase
activity by using GC-MS. NO significant hydroxylase activity could be detected (i.e., less
than 0.5% product formed from 250 uM thymine) after a 1 h incubation.

In conclusion, we have identified two genes encoding T7H-like proteins in T. b.
brucei, demonstrated that the genes are expressed at greater levels in the BF over the PF
stage, shown that both proteins are present in each life stage (possibly with greater
abundance in the PF life stage), and expressed the genes in E. coli. We purified the two
recombinant proteins, demonstrated that they form an Fell/OLKG chromophore (indicating
proper folding), and tested for catalytic activity and DNA binding. Our results provide
no indication of the recombinant proteins being able to hydroxylate free thymine or
thymidine within DNA or to bind DNA. Furthermore, no hydroxylase activity was
detected in extracts from trypanosomes of either life stage. The roles of these proteins in
T. b. brucei remain unknown, but our evidence provides strong arguments against their

participation in pyrimidine salvage.

46

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Borst, P, Sabatini, R. 2008. Base I: discovery, biosynthesis, and possible
functions. Annu. Rev. Microbial. 62: 235-251

48

19.

20.

21.

22.

23.

24.

Ulbert, S, Cross, M, Boorstein, RJ, Teebor, GW, Borst, P. 2002. Expression of the
human DNA glycosylase hSMUGl in Trypanosoma brucei causes DNA damage

- and interferes with J biosynthesis. Nuc. Acids Res. 30: 3919-3926

Gommers-Ampt, J H, Teixeira, AJ, van de Werken, G, van Dijk, WI, Borst, P.
1993. The identification of hydroxymethyluracil in DNA of T rypanosama brucei.
Nuc. Acids Res. 21: 2039-2043

Cross, M, Kieft, R, Sabatini, R, Wilm, M, de Kort, M, van der Marel, GA, van
Boom, J H, van Leeuwen, F, Borst, P. 1999. The modified base J is the target for a
novel DNA-binding protein in kinetoplastid protozoans. EMBO J. 18: 6573-6581

DiPaolo, C, Kieft, R, Cross, M, Sabatini, R. 2005. Regulation of trypanosome
DNA glycosylation by a SW12/SNF2-like protein. Mol. Cell 17: 441-451

Yu, Z, Genest, PA, ter Riet, B, Sweeney, K, DiPaolo, C, Kieft, R, Christodoulou,
E, Perrakis, A, Simmons, JM, Hausinger, RP, van Luenen, HG, Rigden, DJ,
Sabatini, R, Borst, P. 2007. The protein that binds to DNA base J in
trypanosomatids has features of a thymidine hydroxylase. Nuc. Acids Res. 35:
2107-2115

Vainio, S, Genest, PA, ter Riet, B, van Luenen, H, Borst, P. 2009. Evidence that

J-binding protein 2 is a thymidine hydroxylase catalyzing the first step in the
biosynthesis of DNA base J. Mol. Biochem. Parasitol. 164: 157-161

49

CHAPTER 2

APPENDIX

The docking studies were performed by Mike Howard.

50

2A.1. INTRODUCTION

Chapter 2 investigated the possible functions of two trypanosomal thymine
hydroxylase-like proteins, TLP5 and TLP7 from T rypanasoma brucei brucei. These two
peptide sequences are 85% identical to each other and about 30% identical to a fungal
(Rhodatorula glutinis) thymine 7-hydroxylase (T7H), which functions in a pyrimidine
salvage pathway (1,2). Trypanosomes are not known to possess a pyrimidine salvage
pathway, but they have been shown to employ the activity of a thymidine hydroxylase in
the first step of the synthesis of a modified base, B-D-glucosylhydroxymethyldeoxyuracil
or base J, in their nuclear DNA (3,4). Thus, I tested whether TLP5 and TLP7 may be
involved in this nucleotide modification in trypanosomes.

The genes were cloned from the T. b. brucei genome and the proteins were
overproduced in E. coli and purified by column chromatography. The properties of these
recombinant proteins were characterized by several biochemical methods and the
expression of the genes was compared for the bloodstream and procyclic forms of the
protozoan. Much of that work was compiled and published in Experimental Parasitology
(see main body of Chapter 2); the experiments omitted fi'om that publication are
described here. This appendix is organized in a modular fashion. Each module explains

the rationale for that method and describes the procedure, results, and any conclusions

drawn from the experiments.

51

2A.2. OLIGOMERIC STATE DETERMINATION

To determine their native sizes and Oligomeric states, the purified and
concentrated recombinant TLP5 and TLP7 proteins were chromato graphed on a
Superdex®75 size exclusion column and the elution positions were compared to those of
standard proteins (BioRad). The collected fractions also were examined by
polyacrylamide gel electrophoresis using a 3-12% Bis-Tris Blue native gradient gel from
lnvitrogen to verify the major Oligomeric form of both TLP5 and TLP7. Figure 2A.1
shows that multiple forms of TLP5 were present as purified from E. coli. The major peak
in the chromatograrn (panel A) eluted at a position corresponding to 36.6 :1: 4 kDa and
contained a monomer as the predominant form, as seen on the native gel (panel B),
although significant levels of the dimeric or trimeric species also were. present during
electrophoresis, consistent with an equilibrium mixture. The minor feature preceding the
main peak in the chromatograrn corresponded exclusively to a dimer or trimer. Finally,
the first small peak in the chromatograrn appeared to consist of a high molecular mass
contaminant that electrOphoresed as a smear on the gel. These results show that the TLP5
protein is predominantly a monomer as purified, though with some dimer or trimer

present. Similar results were observed with TLP7 (data not shown).

52

Figure 2A.1. Native size and Oligomeric state determination of TLP5. A.
Chromatogram of TLP5 run on a Superdex®75 column and compared with BioRad gel
filtration standards (retention times of y-globulin, 158 kDa; ovalbumin, 44 kDa; and
myoglobin, 17 kDa, shown as inverted triangles above chromatogram). B. Native
polyacrylamide gel showing the Oligomeric state and native size of TLP5. Dashed lines
correspond to those on the chromatogram to indicate the fractions run on the gel.

A.

V V V

 

 

50 55 50 65 70 75 so

 

B.
1236__ . ..
1048—‘ ..
720-— “'7'”
480—- -
242“—
146—'“
66—— ~
20 ‘

 

53

2A.3. HOMOLOGY MODEL

There is no crystal structure for TLP5 or TLP7, nor has the fungal T7H been
structurally characterized. To choose a suitable parent structure for the construction of a
homology model, the protein data bank was searched using the Bioinfo Meta Server
(http://metgbioinfopl) and using the amino acid sequence of TLP5 as a query. Based on
sequence identity to proteins of known structure, anthocyanidin synthase from
Arabidopsis thaliana (PDB 1GP4), with a 33 % identity, was used as a parent structure
and the sequence of TLP5 was mapped directly onto the backbone (C01) atom coordinates
from the crystal structure. Side chains were further modeled into the structure using the
program SCWRL (5). Finally, the iron atom and the aKG were modeled into the
structure based on the coordinates of the anthocyanidin synthase structure containing
those elements. The basic structure of the catalytic core, conserved among all iron/aKG
dioxygenases, was observed in TLP5 (Figure 2A.2 panel A). The facial triad residues
made up of His 200, Asp 202, and His 257 in TLP5 were correctly positioned to
coordinate a metal atom. Further, when the iron atom and the aKG cosubstrate were
modeled into the active site, the coordination distances observed were within the normal
range seen in this family of enzymes (6). In addition, a highly conserved arginine known
to participate in the coordination of the C5 carboxyl group of aKG in other members of
this enzyme family also was present here, at position 266.

The coordinates for the model were provided to undergraduate student Mike
Howard, who submitted them as a protein target to the program Dock Blaster

(http://blaster.docking.org) to identify possible classes of ligands that may fit into the

54

modeled active site (7). A specified active site, using the residues pictured in Figure
2A.3, also was submitted in PDB format. This program used the ZINC database of
commercially available small ligands (8) and docked them into the active site based on
the lowest energy binding modes. Ligand candidates were then scored and ranked by
binding energies. The program identified the three best ligands for the TLP5 homology
model active site, which were all ring-based structures resembling nucleic acids.

The C-terminal a-helix of the TLP5 homology model was predicted to rest against
the putative opening to the active site and appeared poised to act as a gate, limiting access
to the oxygen-activating iron atom (Figure 2A.2 panel B). This model led to two
hypotheses that were experimentally tested. First, the polyhistidine tag, added during the
cloning process to facilitate protein purification, may have interfered with movement of
the C-terminal helix required for activity and would thus explain the lack of activity
observed to that point. Therefore, the purification tag was moved to the N-terrninus and
attempts to characterize the alternatively tagged protein were repeated (subsection 4).
Second, one could postulate that another factor in the trypanosome, but not present in my
in vitro system, could induce movement of the C-terrninal helix and allow activity. To
test this hypothesis, a truncation mutant was created that eliminated the C-terminal 13
residues making up that helix and the corresponding protein was characterized

(subsection 5).

55

Figure 2A.2. Homology model of TLP5. A. Ribbon diagram colored as a spectrum
from the N-terminus in blue to the C-terminus in red. Breaks in the model represent
areas where there was insufficient homology to model. The iron-coordinating facial triad
and aKG-stabilizing arginine are shown as sticks and colored by element (carbon-green,
oxygen-red, nitrogen-blue). The aKG is also shown as sticks and colored by element
(carbon-cyan, oxygen-red). The iron atom is shown as a magenta sphere. B. Surface
representation of the same model colored by element (carbon-white, oxygen-red,

nitrogen-blue, sulfur-yellow). Figures were created using PyMol.

A. B.

 

56

Figure 2A.3. Results of docking a subset of the ZINC small molecule database into the
modeled active site of TLP5. Each panel has a chemical structure and a modeled binding
depiction of a low-energy, docked ligand molecule. TLP5 is shown as magenta lines, the
iron atom as an orange sphere, the aKG as either yellow or purple sticks, and the docked
ligand as blue sticks. Panel C is rotated 60° from the angle of panels A and B to better

show the possible molecular interactions. Binding figures were created using PyMol.

 

57

2A.4. RELOCATION OF PURIFICATION TAG

To investigate whether the C-terminal histidine tag interfered with the activity
assays, the purification tag was moved to the N-terrninus of the protein. The gene
encoding TLP5 was cloned out of its initial expression plasmid, pET28b (Novagen), by
using forward (5’-AGGATATAGC TAGCATGGCT CACGGCTCGA T-3’) and reverse
(5’-GTGGTGGTGC TCGAGTCACA TCT'I‘TGTI‘TT G-3 ’) primers to reintroduce the
stop codon before the C-terminal purification tag and move the start site downstream of a
sequence encoding the N-terrninal polyhistidine purification tag. This process introduced
six extra residues between the purification tag and the initial methionine of the actual
protein sequence. The recombinant protein was overproduced in E. coli and purified as
before (see main body of Chapter 2) though the protein was less stable and precipitated
after even brief storage at 4 °C.

The recombinant, N-terrninally-tagged TLP5 protein was concentrated to about
180 uM and assayed for formation of the diagnostic metal-to-ligand charge-transfer
chromophore in the presence of Fell and OLKG as seen in the original C-terrninally tagged
protein preparation. The spectroscopic assay was performed as described in the main
body of Chapter 2 and no chromophore was observed. This result indicated that the
protein with the extra N-terminal residues did not fold properly and was thus unable to
bind the iron and aKG in the correct orientation. Therefore, the N-terminally tagged

protein was not used for further experiments.

58

2A.5. C-TERMINAL TRUNCATION

The C-terrninal a-helix of TLP5 appeared to occlude access to the active site in
the homology model, fireling the hypothesis that removing those residues may provide
access to the active site in the absence of some required in viva post translational
modification or protein-induced activation. To test this hypothesis, a 13 residue
truncation mutant was created to remove this helix. The gene encoding TLP5 was cloned
out of genomic trypanosome DNA using the original forward primer and the new reverse
primer (5’-GAGCATCCTC GAGAGTCTTC AGCAACCAAT CC—3’) to replace the
sequence encoding the C-terminal 13 residues with a stop codon.

Truncated protein, termed TLP5AC13, was produced in E. coli and purified as
described in the main body of Chapter 2. The shortened protein was concentrated to 65
pM and assayed anaerobically for the characteristic metal-to-ligand charge-transfer
chromophore as before. The feature was present, with an estimated extinction coefficient
of 60 M'1 cm'l indicating that the feature was incompletely formed compared with
previously reported intensities (9). TLP5AC13 also was assayed for enzymatic activity
by measuring oxygen consumption with an oxygen electrode, but no activity was
observed for any of the substrates tested including thymine, thymidine, and oligo polyT.
Finally, the truncated protein was assayed for DNA binding (see subsection 6 for
method). No binding was observed with any of the substrates tested including
supercoiled and linearized plasmids, with or without alkylation, or HindIII lambda DNA
fragments. This protein tended to precipitate during experiments; therefore, further

experiments were not performed with this mutant.

59

2A.6. DNA BINDING

The sequence similarity of TLP5 and TLP7 to the fungal T7H led to the
hypothesis that the trypanosomal proteins may be nucleotide-modifying enzymes. One
possibility is that the substrate is a polynucleotide, therefore the proteins were tested for
their abilities to bind to various DNA substrates via an electrophoretic mobility shift
assay using agarose gels. Briefly, proteins were incubated with DNA and various
supplements for 30 min at 37 °C. Glycerol was added to the samples to 5% final
concentration and they were loaded onto a 0.8 % agarose gel and run in 89 mM Tris
borate buffer. Additives tested in the binding assays included binding buffer (30 mM
imidazole and 10 mM Tris, pH 7.9, containing 150 mM NaCl), Tris buffer (25 mM Tris,
pH 8.0, containing 100 mM KCl and with or without 5 mM MgCl2, 0.5 mM DTT, or 0.2
mM EDTA), or Hepes buffer (50 mM Hepes, pH 8.0, containing 40 uM CoCl2, 100 pM
ascorbate, and 200 uM aKG). Other assay components included in some of the assays
included up to 200 uM uKG, 50 uM F e", and 100 uM ascorbate. DNA substrates
included supercoiled plasmid, linearized plasmid, methylated plasmid, and HindlII A
DNA fragments. No shift in the DNA bands was observed that was greater than the
variation in band positions present in lanes without protein. This result, taken together
with the lack of detectable enzymatic activity challenged the hypothesis that TLP5 and
TLP7 were polynucleotide-modifying enzymes. Therefore, other methods were

employed to look for possible substrates and functions of these proteins.

60

2A.7. IN WVO EPITOPE TAGGING OF THE TLP5 GENE

Data acquired by quantitative reverse transcriptase PCR amplification of T LP5
and T LP7 indicated that the steady state mRNA levels were higher in the bloodstream
form trypanosomes than the procyclic forms. While interesting, the picture remained
incomplete as most of the genetic regulation in trypanosomes takes place at the level of
protein expression and turnover (10). Therefore, a construct was designed to add
sequence coding for a nine residue hemagglutinin (HA) epitope tag to the chromosomal
copy of T LP5. This would allow the analysis of in viva protein levels under various
stress conditions as well as determination of the subcellular localization for the protein,
and it would provide a method to observe changes in the protein production levels in
concert with other procedures such as RNAi.

The initial HA tagging construct was acquired from Professor Donna Koslowsky.
It consisted of the sequence coding for the HA tag, an a/B tubulin spacer region, and a
blasticidin resistance gene. About 100 nucleotides corresponding to the 3’ end of the T LP5
gene were added to the 5’ end of the construct by PCR amplification using the primer (5’-
CCA'I'TCGCCA AGCAATCCCC CCAAATATCC ACCAGTCCGT GCTGTGGATT
GGTTGCTGAA GCGTTTCGCG GAAACATATG CCCATCGCAA AACAAAGATG
TACCCATACG ACGTCCCAGA CTACGCTIAA- 3’) where the stop cation is
underlined. In addition, about 100 nucleotides corresponding to the beginning of the 3’
untranslated region was added to the 3’ end of the construct by PCR amplificationusing the
primer (5’- GAAAAGAAAA AAAAAGAGTT TGAAGACACA "ITCATTCATI’

CATTGCCACC GCATCCGTAC CGACGGGGGC CTAACGAAAA GATTACGTTC

61

AGTTATTGAT ILAGCCCTCC CACACATAAC CAGAG -3’) where the reverse
complement of the stop codon for the blasticidin resistance gene is underlined. This first
round of PCR amplification created the unique tagging construct needed for my tagging
procedure. Next, this unique construct was amplified by a second round of PCR using the
forward primer (5’- CCATTCGCCA AGCAATCCCC CCAAA -3 ’) and the reverse primer
(5’- GAAAAGAAAA AAAAAGAG'IT TGAAGACACA TTCATTCATT -3 ’) (Figure
2A.4). The PCR products were separated from unused reagents by using the Qiagen PCR
clean-up kit and used directly for trypanosome transfections.

In replicate trials, 2.5 or 5 pg of the DNA-tagging construct were added to 108
procyclic trypanosome cells in 0.5 mL phosphate-buffered sucrose in a BioRad
electroporation cuvette. Cells were treated in a BioRad electroporator at 1.5 kV, 25 uF,
and infinite resistance for two pulses Spaced by 10 sec. Cells were transferred immediately
to a small culture flask containing 4.5 mL of SDM-79 medium with 10 % fetal bovine
serum and 7.5 ug/mL hemin (as an iron source) along with penicillin and streptomycin to
select against bacteria. Cultures were allowed to grow at 27 °C overnight with gentle
shaking before adding the eukaryotic selection drugs: 15 ug/mL neomycin (to maintain the
tet repressor), 100 ug/mL hygromycin (to maintain the T7 polymerase), and 10 ug/mL
blasticidin (to maintain our tagging construct). Cells were grown for an additional two
days and then serially diluted in the same selective media to create a more clonal
population. One of the dilution cultures was grown for approximately a week before some
of the cells were harvested and genomic DNA extracted to test the incorporation of the

tagging construct.

62

The entire T LP5 gene segment of chromosome 5 was amplified by PCR using the
forward primer (5’- GCCATTTTT CTGTGAGCCT AATCC -3 ’) and the reverse primer
(5’- TCAGG ACAGATACGA GCAAAAGAAG-3’). With the incorporated tag, the
lengths of the products would be a mixture of approximately 1 kb for the tagged allele
and 300 bases for the untagged allele, since trypanosomes are diploid. In addition, the
segment was amplified by using primers internal to the tagging sequence that would
further test its placement when paired with the primers described above; these hybridize
to regions just upstream and downstream of the sequences included in the original
unique-tagging construct. The internal primers corresponded to a forward segment near
the end of the blasticidin resistance gene (5’-GGGATTCGTGAATTGCTGCCCTCT-3’)
and a reverse segment (5’-TGTACCACGCTGCAACAGTGTGAAAATTCGA-3’) near
the beginning of the 01/0 tubulin spacer (Figure 2A.4).

One culture out of six separate tagging experiments, each with six transfections,
appeared to have the correct tag incorporation in at least one allele as seen by the PCR
verification. Therefore, a whole cell lysate was made from a sample of that culture and
was Western blotted using the anti-HA antibody conjugated to alkaline phosphatase as a
probe. Briefly, 107 cells were harvested by centrifugation and resuspended in 1 mL cell
wash buffer (20 mM Tris, pH 7.5, containing 100 mM NaCl and 3 mM MgCl2) and placed
in a bath sonicator with ice for about 20 min. Debris was removed by centrifugation at
16,000 g for about 10 min. The supernatant was electrOphoresed on a denaturing 12%
polyacrylamide gel, proteins were transferred to an ImmobilinP PVDF membrane.
(Millipore), and the membrane was probed with rabbit anti-HA antibody from Sigma.

Unfortunately, no protein band of the appropriate size was detected. Several high

63

molecular weight bands were detected that are commonly seen in blots of trypanosome

extracts probed with the anti-HA antibody (Donna Koslowsky personal communication).

64

Figure 2A.4. Hemagglutinin epitope tagging of T LP5. This figure is a construction
schematic where the nine residue hemagglutinin (HA) epitope is shown in green, the a/B
tubulin spacer in yellow, and the blasticidin resistance gene in blue. The 3’ end of the

T LP5 gene is shown in red and the 3’ untranslated region in brown as parts of the primers
used to make the unique tagging construct by PCR. This construct was amplified by a 2°
PCR reaction using primers whose positions are indicated by the red and brown lines
above the scheme. Finally, primers whose positions are indicated by the red and brown
open boxes and the grey arrows were used to test the incorporation of the tag into

chromosome 5 of the trypanosome.

 

 

1 =-
Tx‘q- A ‘0‘?
. ‘3‘! ’3:
“a. ._,
01/0 Tubulin —__ _ _ _B,SR . _
YPYDVPDYA l1° PCR
. 2° PCR
, :; .. . . 3 m '-
- HA}- a/p Tubulin -_ BSR :3
+— -——->

65

2A.8. PULL-DOWN ASSAYS WITH TLP5 AND TRYPANOSOME CELL

EXTRACT

All attempts to detect activity with various nucleic acid substrates failed,
therefore pull down assays with trypanosome cell extracts were used in an effort to find
potential protein interaction partners of TLP5. E. coli cells over-expressing TLP5 were
lysed, sonicated, and ultracentrifuged to generate soluble cell extracts. This sample was
added to Ni-NTA cellulose beads and the histidine tagged TLP5 allowed to bind to the
resin. The
beads were washed with binding buffer twice and then trypanosome soluble cell extracts
(prepared as before) were added and incubated at room temperature for 20 min to allow
binding. Again, the resin was washed with binding buffer and eluted in elution buffer
(150 mM imidazole and 10 mM Tris, pH 7.9, containing 150 mM NaCl). Extract, wash,
and elution samples were electrOphoresed on a denaturing 12% polyacrylamide gel and
silver stained to detect proteins of low abundance.

The gel lanes were compared as described above for E. coli extracts, T. b. brucei
extracts, and mixtures of the extracts. With three separate experiments, including
analyses using free resin in an eppendorf centrifuge tube and resin packed into a small
column, no new bands were identified in the combined extract runs that were not also
present in the control runs. Thus, the pull-down experiments failed to identify potential

interaction partners of TLP5.

66

2A.9. CONCLUSIONS

The thymine hydroxylase-like proteins TLP5 and TLP7 from T. b. brucei have
been characterized and determined to be members of the non-heme iron and aKG
dependent dioxygenase superfamily of enzymes. Extensive efforts were undertaken to
identify the activity of these proteins, though none of the tested hypotheses were
supported. Under the conditions used, no DNA binding was observed and no activity
was demonstrated using various substrates, even when examining modified proteins to
eliminate possible active site access restrictions. Pull-down assays failed to identify
protein interaction partners and docking studies did not yield obvious ligand choices to
pursue. While the true purpose of these proteins in the cell remains enigmatic, they
probably do not participate in polynucleotide modification but are very likely to catalyze

some other type of hydroxylation reaction in the cells.

67

10.

REFERENCES

Smiley, JA, Kundracik, M, Landfried, DA, Barnes, VR, Sr., Axhemi, AA. 2005.
Genes of the thymidine salvage pathway: thymine-7-hydroxylase from a
Rhodatorula glutinis cDNA library and iso-orotate decarboxylase from
Neurospora crassa. Biochim Biophys Acta 1723: 256-264

Neidigh, JW, Darwanto, A, Williams, AA, Wall, NR, Sowers, LC. 2009. Cloning
and characterization of Rhodatorula glutinis thymine hydroxylase. Chem Res
T oxicol 22: 885-893

Gommers-Ampt, JH, Van Leeuwen, F, de Beer, AL, Vliegenthart, JF, Dizdaroglu,
M, Kowalak, JA, Crain, PF, Borst, P. 1993. beta-D-glucosyl-
hydroxymethyluracil: a novel modified base present in the DNA of the parasitic
protozoan T. brucei. Cell 75: 1129-1136

Gommers-Ampt, J H, Teixeira, AJ, van de Werken, G, van Dijk, WJ, Borst, P.
1993. The identification of hydroxymethyluracil in DNA of T rypanosoma brucei.
Nucleic Acids Res 21 : 2039-2043

Canutescu, AA, Shelenkov, AA, Dunbrack, RL, Jr. 2003. A graph-theory
algorithm for rapid protein side-chain prediction. Protein Sci 12: 2001-2014

Hausinger, RP. 2004. Fe(II)/or-ketoglutarate-dependent hydroxylases and related
enzymes. Crit. Rev. Biochem. Mal. Biol. 39: 21-68

Irwin, JJ, Shoichet, BK, Mysinger, MM, Huang, N, Colizzi, F, Wassam, P, Cao,
Y. 2009. Automated docking screens: a feasibility study. J Med Chem 52: 5712-
5720

Irwin, IJ, Shoichet, BK. 2005. ZINC-~a free database of commercially available
compounds for virtual screening. J Chem Inf Model 45: 177-182

Ryle, MJ, Padmakumar, R, Hausinger, RP. 1999. Stopped-flow kinetic analysis of
Escherichia coli taurine/oc-ketoglutarate dioxygenase: interactions with or-

ketoglutarate, taurine, and oxygen. Biochemistry 38: 15278-15286

Clayton, C, Shapira, M. 2007. Post-transcriptional regulation of gene expression
in trypanosomes and leishmanias. Mol Biochem Parasitol 156: 93-101

68

CHAPTER 3

SEARCH FOR HYDROXYLASE ACTIVITY IN THE TRYPANOSOMAL

PROTEIN, JBPl

Portions of these studies were published along with experiments conducted by others in:

Yu, Z, Genest, PA, ter Riet, B, Sweeney, K, DiPaolo, C, Kieft, R, Christodoulou, E,
Perrakis, A, Simmons, JM, Hausinger, RP, van Luenen, HG, Rigden, DJ, SabatiniR,
Borst, P. 2007. The protein that binds to DNA base J in trypanosomatids has features of a
thymidine hydroxylase. Nuc. Acids Res. 35: 2107-21 15

The introduction for this chapter mentions some of the other studies, citing this reference,
as well as prior background and more recent results by others.

69

3.1 INTRODUCTION

Trypanosomes are eukaryotic kinetoplastid flagellates and the causative agents of
several neurological diseases affecting mammals. Chagas Disease, caused by
T rypanosama cruzi and transmitted by redurid bugs, leishmaniasis, associated with
various strains of Leishmania and transmitted by sandflies, and African Sleeping
Sickness, caused by T rypanosoma brucei and carried by the tsetse fly are examples in
humans (1,2). About one percent of the thymine in the telomeric repeats of nuclear DNA
in the mammalian bloodstream form (BF) of T. brucei is replaced with B-D-glucosyl-
hydroxymethyldeoxyuracil (base J) (3 ,4). Even smaller amounts of the modification are
found in subtelomeric regions and very little in chromosome internal locations. This
modified base has several possible functions. First, it is proposed to induce chromatin
compaction and thereby stabilize the chromosome from rearrangements within these
repetitive sequences (5 ,6). Second, base J has a proposed role in the process of antigenic
variation characteristic of T. brucei and other related parasites. Base J is found
throughout the silent variable surface glycoprotein (VSG) expression Sites and the
flanking repetitive sequences, but no I is present in the one active expression site,
suggesting a specific role for J in gene silencing (7). Finally, base I has been identified in
the chromosomal internal polymerase II polycistronic transcription unit strand switch
regions of several kinetoplastids, including T. brucei and L. major, as well as the spacer
regions between individual open reading frames, suggesting a role for base J in the wider
family of kinetoplastids that do not all undergo antigenic variation (8).

Base J is synthesized in two steps. First, the methyl group on the C5 position of a

thymidine already incorporated into DNA must be hydroxylated to create the

70

intermediate hydroxyrnethyldeoxyuridine (HOMedU) (Figure 3.1 panel A), as evidenced
by the presence of low levels of HOMedU in BF trypanosome DNA (9). Next, the
hydroxymethyl moiety must be glucosylated. This reaction is not specific to the
sequence or life cycle-stage as evidenced by the random placement of base I in the
bloodstream and procyclic forms of cells grown on HOMedU (5).

Recently, a protein was identified in T. brucei that specifically binds to base I in
DNA (10). This I binding protein 1 (JBPI) is ~90 kDa protein whose N terminal domain
shows some sequence Similarity to other non-heme iron and a-ketoglutarate (aKG)
dependent hydroxylases by manual alignment (1]). Secondary structure prediction
further places this protein in the double-stranded beta helix or “jelly-roll” core fold
family of proteins (11). This homology and the obvious firnctional similarity to the
methylated DNA hydroxylase AlkB (a DNA repair enzyme) and the fungal thymine-7-
hydroxylase, which is involved in the pyrimidine salvage pathway and acts only on the
free base, fueled the hypothesis that JBPl may be the thymine hydroxylase in
trypanosomes.

JBP2, identified in silico based on its sequence identity to JBPl, does not bind to
J -containing DNA, but does possess a SWI-2/SNF-2 chromatin remodeling domain at its
C—terminal end and is capable of inducing de novo base J synthesis when expressed in
procyclic T. brucei cells (12,13). These cells normally do not express JBPl or JBP2 and
lack base I (3). JBPl is suggested to maintain 1 levels by stimulating additional J
synthesis in nearby sequences while cells containing modified DNA, but lacking JBPl ,
lose base J by dilution during replication (14). Further work to establish the roles of

these proteins has shown that a JBPl knockout in Crithidiafasciculata (a non-pathogenic

71

Figure 3.1. Synthesis of base J. A. Scheme showing the two step molecular synthesis of
base J. The R group represents the continuing strands of DNA. B. Cartoon depicting the
proposed chromatin specific introduction and maintenance of base J. J BP2 is responsible
for the initial introduction of telomeric and subtelomeric base I, while JBPl is
responsible for the initial introduction of chromosome internal base I (8). JBPl then
binds to these regions and stimulates amplification of the modified base (15). GT-

glucosyl transferase. Figure modified from (1 l).

A: NH aKG succinate H NH
o co A
N 2 2 N
R O , I R O
JBP1/JBP2
" ll
0R containing Fe OR
dT HMdU
Glucose
B-glucosyl
transferase
O
Ho/yé/O/TLNH
H OH
o NAG
OR
dJ

     

strain) reduces J levels to 5% of the wild-type cells (14), a JBP2 knockout reduces base J
levels to 20% of the wild-type cells in studies involving T. brucei and L. tarentolae
(12,16), and a double knockout creates a J-null cell line in T. brucei (8,15). Surprisingly,
both J BPl and JBP2 proteins are dispensable in T. brucei with no phenotypic or growth
effects (8,15), while JBP 1 is essential in L. tarentolae ( l 7) and JBP2 is not (16). Finally,
it has been shown that mutation of the key conserved residues making up the “facial

tria ” of the active site or the conserved arginine believed to stabilize the aKG

cosubstrate in these proteins abolishes their ability to stimulate base J synthesis

(1 1,15,16).

Understanding the enzymes involved in J biosynthesis may lead to the
development of drugs to treat trypanosomiases, as this modified base is found only in the
kinetoplastid flagellates. Yet, no direct in vitro hydroxylase activity has been detected
for either JBPI or J BP2. Therefore, this study sought to demonstrate enzymatic or
uncoupled turnover of J BPI. To test JBPl for aKG dependent hydroxylase activity, I
used recombinant protein with in vitro assays to look for product formation and substrate
consumption. Other members of this enzyme family have been shown to decarboxylate
aKG and consume oxygen in the absence of their primary substrate, termed uncoupled
turnover. These studies primarily focused on the uncoupled reactions of JBPl due to the
lack of sufficient amounts of primary substrate, genomic trypanosome DNA containing
base J. Some putative alternate substrates were tested in attempts to measure the coupled

turnover or stimulate uncoupled turnover.

73

3.2 MATERIALS AND METHODS

3.2.] Determination of succinate production as a measure of uncoupled enzymatic
turnover.

Purified recombinant JBPI was supplied by Henri van Luenen of Piet Borst’s lab
at The Netherlands Cancer Institute in Amsterdam. JBPl was from C. fasciculata (not
pathogenic to mammals) or Leishmania tarentolae in wild type or mutated forms. One
mL reactions in 13 x 100 glass tubes, to provide for efficient aeration, were incubated at
either 30 or 37 °C in either 25 mM Tris or 50 mM Hepes buffer, with the pH ranging
from 7.0 to 8.5. Conditions tested included assays containing ferrous iron (0.05-1 mM),
ascorbate (0.05-5 mM), aKG (0.5-20 mM), NaCl (20 mM), MgCl2 (0.5-10 mM), KCl
(IO-100 mM), EDTA(0 or 20 uM), glycerol (0 or 5%), or Triton X-100 (0 or 0.02%), and
as potential substrates, thymidine (20 mM), DNA oli go containing base J (20 uM), and
genomic trypanosome DNA (40 uM).

Aliquots of 300 pL were taken at various time points and quenched with 5 uL of 6
M sulfuric acid. Samples were centrifuged at 16,000 g for 5 min to remove any protein.
250 uL was loaded onto a 0.45 pm cutoff spin filter and centrifuged. 200 uL of the flow
through fraction was loaded onto an Aminex HPX-87H ion exchange organic acids
column (BioRad) (300 mm x 7.8 mm) and analyzed by high performance liquid
chromatography (HPLC). Organic acids were eluted with 0.013 M H2304 as the mobile
phase and detected by using a Waters refractive index detector set at 35 °C and a ,

sensitivity level of 16. The standards of aKG and succinate eluted at 19.8 and 26.6 min,

74

Figure 3.2. aKG and succinate detection by HPLC. A. A representative chromatogram
showing the retention times for aKG (19.8 min) and succinate (26.6 min). B. Standard
curve showing the detection of succinate down to a lower limit of 5 uM in the injected
volume. The R2 value was 0.9999 and the fitted line was used to determine the amount

of succinate produced in an assay based on the integrated area of the detected peak.

 

 

 

 

A.
25.002
20004
I
15.0041
> 1
2 1000-;
,
1
coo—lfrfrrrrrrT'rrTurrrunrfirr TTT 'Tu . ' ‘ ' ltf’ffif'l ' TTTFTTTTTTTT r-TTTT
19.00 20.00 21.00 22.00 23.00 24.00 25.00 20.00 27.00 28.00 29.00 30.00
Retention time (min)
B.
A 400 -
8
o 350 T
I?
v 300 4
8
I- 250 a
<
.K
a 200 -
01
°- 150 1
8
*6 100 1
a
a, 50 .
‘E
-' 0 m T T l_— LL;
0 50 100 150 200 250

Succinate (pM)

75

respectively, and succinate was detectable to a lower limit of 1 nmol or 5 uM in the

injected volume (Figure 3.2).

3. 2.2 Oxygen consumption as a measure of uncoupled enzymatic turnover.

Assays were performed by using a micro oxygen electrode in an Instech chamber
coupled to a circulating water bath to maintain a temperature of 33° C and a Diamond
Electra-Tech chemical microsensor. Percent oxygen saturation was continuously
monitored throughout the assay. The upper and lower saturation limits were set by
flushing the system with buffer (25 mM imidazole, pH 7.0) that was bubbled with air or
degassed buffer bubbled with nitrogen, respectively.

JBPl, as received from the Netherlands, was in 25 mM Hepes, pH 7.0, with 150
mM NaCl and 1 mM EDTA. A portion of the protein was dialyzed against 25 mM
imidazole, pH 7.0, and 150 mM NaCl to remove the EDTA. JBPl with or without EDTA
was assayed at 1 uM using variations of the standard conditions: 50 uM ferrous iron, 1
mM aKG, 400 uM ascorbate, 6.5 mM NaCl, and 1 mM of either thymine, thymidine, or
oligo polyT. Assays were carried out in 25 mM imidazole, pH 7.0, in the micro-oxygen

electrode with an assay volume of 600 uL.

3.2.3 Spectroscopic detection of a Metal-to-Ligand Charge- Transfer (MLC T) Feature.
UV/visible spectra were obtained by using an HP 8453 spectrophotometer
(Hewlett Packard) equipped with a circulating water bath. Stock solutions of 10 mM Fe“

and 5 mM 01KG were made by adding the dry reagents to vials, subjecting them to

repeated cycles of vacuum and argon, and adding anaerobic water by using a syringe.

76

The JBPl protein was provided in a high salt buffer with a reducing agent for shipping,
therefore to prevent possible interference in these experiments it was first exchanged (at 4
°C by repeating concentration/dilution by using an Amicon double-membrane centrifiigal
concentrator with a 10 kDa cutofl) into 25 mM Tris, pH 8.0, containing 50 mM KCl. 50
uM protein was added to a l—cm path length, 300 uL, black-walled cuvette and made
anaerobic by the same method, followed by scanning at 9 °C. Four equivalents of aKG
were added followed by a titration of one equivalent of Fe" into the cuvette in 0.2
equivalent increments with scans after each addition. Data were analyzed by using the
software IGOR6. First, data were corrected for baseline shifts arising from cuvette
repositioning to have a uniform absorbance at 850 nm. Then, data points over a 5 nm
range of wavelengths on each side of every point were averaged to smooth the data and

the wavelength maxima of the observed features were determined by taking the second

derivative.

77

3.3 RESULTS AND DISCUSSION

The trypanosome protein J BPl is known to bind to J -containing DNA and has
been implicated in the process of antigenic variation and chromatin maintenance. It was
hypothesized that the C-terminal domain functions in J base recognition and binding
while the N-terrninal domain serves as a non-heme iron and aKG-dependent hydroxylase
responsible for the initial step in the Synthesis of newly modified bases in adjacent DNA
regions. If this is the case, then oxygen and aKG would be consumed in the reaction and
succinate would be produced.

Assays for oxygen consumption and aKG decomposition were performed by
using an oxygen electrode and HPLC, respectively, with purified recombinant JBPl that
was known to bind DNA oligos containing base J (as evidenced by gel band Shifts).
Methods included assays for both coupled and uncoupled turnover. The assay was
verified using taurine hydroxylase. Various nucleic acid substrates were tested, though
the amounts available were severely limiting. None of the assays analyzed by oxygen
electrode exhibited oxygen consumption greater than the background levels measured in
the absence of protein. The background oxygen consumption is thought to arise from the
oxidation of ascorbate in the presence of iron (18). In a similar manner, none of the
assays analyzed by HPLC Showed appreciable production of succinate over a basal level
that was measurable when no protein was present. This background amount of succinate
is thought to arise from the spontaneous breakdown of OLKG in the presence of iron,

oxygen, and ascorbate.

78

To confirm proper folding of J BPl , the purified recombinant protein was
examined for its ability to form a characteristic MLCT chromophore seen in other
members of the Fen/(IKG-dependent dioxygenases and associated with binding of the
iron cofactor and the aKG cosubstrate (19). The protein was monitored by UV-visible
spectroscopy under anaerobic conditions, using low temperature to maintain stability,
while titrating in ocKG and Fe". As shown in Figure 3.3, anaerobic JBPl generated the
diagnostic MLCT transition at 504 nm when both metal and cofactor were present. Due
to the light scattering underneath the feature, an extinction coefficient was not estimated
from this data.

While J BPl and J BP2 have been shown by mutagenesis studies to be responsible
for the specific introduction of base J into kinetoplastid DNA, in vitro activity has not
been directly detected for either protein. JBPl purified from recombinant cells was
shown to be properly folded and capable of binding iron and aKG in a manner that
formed a characteristic MLCT spectrum. This result verifies the protein’s membership in
the non-heme iron and aKG dependent dioxygenase superfamily of enzymes. Since the
activity assays performed here were unsuccessful, it is possible that the substrate for the
J BPl enzyme may be a Specific form of chromatin that is recognized by its structure
rather than sequence. Therefore, even the genomic trypanosome DNA containing base J,

would not elicit enzymatic turnover under the conditions we tested.

79

Figure 3.3. Spectroscopic evidence for binding of F e" and aKG by JBPl. The anaerobic
UV/visible spetrum of J BPl (50 uM in 25 mM Tris, pH 8.0 containing 50 mM KCl) was
examined for the sample as prepared, after adding 200 uM aKG, and while titrating in
Fe]I with stirring at 9 °C. The (Fell-aKG-protein minus aKG-protein) difference spectra
shown correspond to the addition of 0 (baseline), 0.2, 0.4, 0.6, 0.8, and 1 equivalent of

metal ions.

0.16
0.14
0.12
0.10 "

0.08 d

A Absorbance

0.06 -

I

II/

0.04 a

0.02 b
. K-n...

ODD-4r T I """"*‘“* W '

I
400 500 600 700 800
wavelength (nm)

 

 

 

 

80

10.

11.

REFERENCES

Barrett, MP, Burchmore, RJ, Stich, A, Lazzari, J O, Frasch, AC, Cazzulo, JJ,
Krishna, S. 2003. The trypanosomiases. Lancet 362: 1469-1480

Herwaldt, BL. 1999. Leishmaniasis. Lancet 354: 1191-1199

Gommers-Ampt, J H, Van Leeuwen, F, de Beer, AL, Vliegenthart, J F, Dizdaroglu,
M, Kowalak, JA, Crain, PF, Borst, P. 1993. beta-D-glucosyl-
hydroxymethyluracil: a novel modified base present in the DNA of the parasitic
protozoan T. brucei. Cell 75: 1129-1136

van Leeuwen, F, Dirks—Mulder, A, Dirks, RW, Borst, P, Gibson, W. 1998. The
modified DNA base beta—D-glucosyl-hydroxymethyluracil is not found in the
tsetse fly stages of Trypanosoma brucei. Mol Biochem Parasitol 94: 127-130

van Leeuwen, F, Kiefi, R, Cross, M, Borst, P. 1998. Biosynthesis and function of
the modified DNA base betavD-glucosyl-hydroxymethyluracil in Trypanosoma
brucei. Mol Cell Biol 18: 5643-5651

van Leeuwen, F, Kiefi, R, Cross, M, Borst, P. 2000. Tandemly repeated DNA is a
target for the partial replacement of thymine by beta-D-glucosyl-
hydroxymethyluracil in Trypanosoma brucei. Mol Biochem Parasitol 109: 133-
145

van Leeuwen, F, Wijsman, ER, Kiefi, R, van der Mare], GA, van Boom, J H,
Borst, P. 1997. Localization of the modified base J in telomeric VSG gene
expression sites of Trypanosoma brucei. Genes Dev 11: 3232-3241

Cliffe, LJ, Siegel, TN, Marshall, M, Cross, GA, Sabatini, R. 2010. Two thymidine
hydroxylases differentially regulate the formation of glucosylated DNA at regions
flanking polymerase II polycistronic transcription units throughout the genome of
Trypanosoma brucei. Nucleic Acids Res 38: 3923-3 935

Gommers-Ampt, J H, Teixeira, AJ, van de Werken, G, van Dijk, WJ, Borst, P.
1993. The identification of hydroxymethyluracil in DNA of Trypanosoma brucei.
Nucleic Acids Res 21 : 2039-2043

Cross, M, Kiefi, R, Sabatini, R, Wilm, M, de Kort, M, van der Marel, GA, van
Boom, J H, van Leeuwen, F, Borst, P. 1999. The modified base J is the target for a
novel DNA-binding protein in kinetoplastid protozoans. EMBO J 18: 6573-6581

Yu, Z, Genest, PA, ter Riet, B, Sweeney, K, DiPaolo, C, Kieft, R, Christodoulou,
E, Perrakis, A, Simmons, JM, Hausinger, RP, van Luenen, HG, Rigden, DJ,

81

12.

l3.

14.

15.

16.

17.

18.

19.

Sabatini, R, Borst, P. 2007. The protein that binds to DNA base J in
trypanosomatids has features of a thymidine hydroxylase. Nucleic Acids Res 35 :
2107-2115

DiPaolo, C, Kiefi, R, Cross, M, Sabatini, R. 2005. Regulation of trypanosome
DNA glycosylation by a SWIZ/SNFZ-like protein. Mol Cell 17: 441—451

Kieft, R, Brand, V, Ekanayake, DK, Sweeney, K, DiPaolo, C, Reznikoff, WS,
Sabatini, R. 2007. J BP2, a SW12/SNF2-like protein, regulates de novo telomeric

DNA glycosylation in bloodstream form Trypanosoma brucei. Mol Biochem
Parasitol 156: 24-31

Cross, M, Kiefi, R, Sabatini, R, Dirks-Mulder, A, Chaves, I, Borst, P. 2002. J -
binding protein increases the level and retention of the unusual base J in
trypanosome DNA. Mol Microbiol 46: 37-47

Cliffe, LJ, Kieft, R, Southern, T, Birkeland, SR, Marshall, M, Sweeney, K,
Sabatini, R. 2009. JBPl and J BP2 are two distinct thymidine hydroxylases

involved in J biosynthesis in genomic DNA of African trypanosomes. Nucleic
Acids Res 37: 1452-1462

Vainio, S, Genest, PA, ter Riet, B, van Luenen, H, Borst, P. 2009. Evidence that
J -binding protein 2 is a thymidine hydroxylase catalyzing the first step in the
biosynthesis of DNA base J. Mol Biochem Parasitol 164: 157-161

Genest, PA, ter Riet, B, Dumas, C, Papadopoulou, B, van Luenen, HG, Borst, P.
2005. F orrnation of linear inverted repeat amplicons following targeting of an
essential gene in Leishmania. Nucleic Acids Res 33: 1699—1709

Klein, SM, Cohen, G, Cederbaum, A1. 1981. Production of formaldehyde during
metabolism of dimethyl sulfoxide by hydroxyl radical generating systems.
Biochemistry 20: 6006-6012

Ryle, MJ, Padmakumar, R, Hausinger, RP. 1999. Stopped-flow kinetic analysis of

Escherichia coli taurine/or-ketoglutarate dioxygenase: interactions with (X.-
ketoglutarate, taurine, and oxygen. Biochemistry 38: 15278-15286

82

 

CHAPTER 4

CHARACTERIZATION OF A T RYPANOSOMA BRUCE] ALKB HOMOLOG

CAPABLE OF REPAIRING ALKYLATED DNA

Portions of the introduction to this chapter were published in:
Simmons, J M, Muller, TA, Hausinger, RP. 2008. Fe(II)/alpha-ketoglutarate hydroxylases

involved in nucleobase, nucleoside, nucleotide, and chromatin metabolism. Dalton Trans.
5132-5142.

83

 

ABSTRACT

T rypanosoma brucei encodes a protein homologous to AlkB of Escherichia coli,
raising the possibility that trypanosomes catalyze oxidative repair of alkylation-damaged
DNA. The gene encoding the T. brucei AlkB homolog (TbABH) was cloned and
expressed in E. coli, the recombinant protein was purified and characterized, and the
capacity of the protozoa] gene to complement alkB bacteria cells was examined. When
anaerobic TbABH was incubated with Fe" and a-ketoglutarate (orKG), the sample
produced a characteristic metal-to-ligand charge-transfer chromophore, confirming its
membership in the Fell/aKG dioxygenase superfamily. TbABH was Shown to bind to
DNA, exhibiting a clear preference for binding to alkylated DNA according to results
derived by electrophoretic mobility shift assays. Further, TbABH partially
complemented an alkB knockout in E. coli cells stressed with methylmethanesulfonate,

confirming the assignment of TbABH as a functional AlkB protein in T. brucei.

84

4.1 INTRODUCTION

Trypanosomes are eukaryotic extracellular parasites that cause diseases in various
mammalian hosts (1). They are evolutionarily divergent organisms that branched early
from the phylogenetic tree, before fungi, plants, or animals (2). Trypanosoma brucei, the
causative agent of African Sleeping Sickness in humans, has a dense exterior protein coat
that limits access to the parasite’s cellular membrane (3,4). In bloodstream form cells,
the coat protein is expressed from a single allele and changes periodically in a population
to evade detection by the host immune system; a process called antigenic variation (5).
The genes encoding the repertoire of coat proteins are located primarily in the
subtelomeric regions among large patches of repetitive sequence and are relocated by
homologous recombination into a promoter-containing expression site as needed (6,7).

The many repetitive regions in the T. brucei genome and the high frequency of
recombination events lead to the need for efficient DNA repair and maintenance
mechanisms. Components for most of the common DNA repair pathways have been
identified in trypanosomes and some have been characterized. Proteins homologous to
certain elements of the mammalian mismatch repair pathway (8), base excision repair (9),
nucleotide excision repair (10) and homologous recombination (7) are present. One
method of DNA repair not yet described in trypanosomes is direct repair which can be
catalyzed by several DNA glycosylases, alkyl transferases, and hydroxylating enzymes in

other systems. One such hydroxylase is AlkB, which has been best characterized in

Escherichia coli.

85

E. coli alkB has been studied for its role in the adaptive response to alkylation
damage since the 19808 (11,12). This gene was long known to confer resistance to
certain methylating agents, and in 2002 the encoded protein was discovered to be a
member of the F ell/a-ketoglutarate (aKG) dioxygenases (13,14). The enzyme catalyzes
N-dealkylation of l-methyladenine (lmeA) and 3-methylcytosine (3meC) in DNA by the
mechanism shown in Figure 4.1, creating an unstable intermediate that spontaneously
releases an aldehyde to regenerate the native base. AlkB repairs the analogous lesions in
RNA (15), including mRNA and tRNA (16). Furthermore, the protein dealkylates 1-
methylguanine, 3-methylthymine and several etheno adducts of DNA (17-20).

The crystal structure of E. coli AlkB in complex with F e", OLKG, and the tri-
deoxynucleotide substrate T-lmeA-T (Fig. 4.2) reveals details of the metallocenteri active
site and helps to clarify how the enzyme can accommodate its various substrates that
include single-strand (ss)-DNA, double-strand (ds)—DNA, and various forms of RNA
(21). Residues His 131, Asp 133, and His 187 bind the metal, aKG coordinates Fe" in a
bidentate manner (via the C1 carboxylate and C2 keto groups), and a salt bridge from Arg
204 stabilizes the aKG C5 carboxylate. The open coordination site for oxygen binding
does not point toward the substrate (a situation termed “off-line” geometry) (22); thus
requiring a Shift in position of the C1 carboxylate prior to oxygen binding or a “ferryl
flip” to occur after generating the F e'v—oxo intermediate (23). The trimer substrate is bent
to extend the damaged base deep into the active-site pocket, making ten identified

hydrogen bonds and many more van der Waals contacts with the protein.

86

Figure 4.1. Reactions of AlkB with lmeA and 3meC in DNA or RNA.

NHZ NHz

N + N
</ l \ N/ </' )NI‘SJ </ f N
N N/l ; : /l_—T_> N N2
02 C02 HCHO

DNA or RNA aKG succinate DNA or RNA DNA or RNA

NH2 OH (lib.

002 HCHO
DNA or RNA GOKG 911990319 DNA 0, RNA DNA or RNA

87

 

Figure 4.2. Structure of AlkB (PDB 2fd8). The left panel depicts the overall fold
of the enzyme, while the right panel highlights the active site residues. Fe" is an orange
sphere, aKG has yellow carbon atoms, metal ligands have blue carbon atoms, a cofactor-
stabilizing residue is shown with orange carbon atoms, and other postulated active site
residues are indicated with white carbon atoms. The bound substrate is shown with
magenta carbon atoms (abbreviated in the right panel). The nucleotide-binding lid
(residues 45-90) is comprised of three B-strands (the left-most strand in the pair of 8-
sheets and two short B-strands at the bottom) along with several loops (bottom left)

lacking well-defined secondary structures.

 

88

The AlkB N-terminus is responsible for about half of the identified contacts with
the substrate via a highly flexible nucleotide recognition domain or lid made up of 3 B-
strands (residues 45-90). This protein region is flexible based upon the superimposed
structures of the bound and nucleotide-free states, thus allowing movement and
reorientation without losing critical contacts. Substrate specificity at the base level is
provided deep in the pocket; a hydrogen bond between Arg 210 and the adenine moiety
could also be made with cytosine, but not with the other two bases, explaining why AlkB
prefers lmeA and 3meC as substrates. Finally, AlkB’s modest preference for SS-
DNA/RN A substrates (15,24) is explained by the nucleotide polymer being oriented to
extend into a hydrophobic pocket with stabilization provided by base stacking with
nearby aromatic residues. By contrast, ds—DNA would need to unwind partially in order
to allow the section of DNA containing the lesion to extend into the active site.

Kinetic studies have examined AlkB activity using a wide range of substrates. A
rate of 12 min'1 was reported for randomly methylated poly(dA) (25), or about 4 min’1 for
two specifically mono-methylated oligonucleotides (26). Catalytic rates with the tri-
deoxynucleotide T-lmeA-T used in the crystal structure studies were determined
separately by two groups and found to be 7.4 and 4.5 min", respectively (21,25). A
phosphate 5’ of the methylated base is critical for proper recognition based upon a 6-fold
increase in the apparenth when it is missing (25), and this result is compatible with the
crystal structure which shows both a hydrogen bond and a salt bridge involving this 5’
phosphate (2 1).

In addition to the dealkylation reactions observed with the above substrates, AlkB

is known to exhibit additional types of reactivity. The minimal substrate that could be

89

demethylated was identified as lme-dAMP (25), whereas the free nucleosides lmeA,
lme-2’-dA, 3meC, and 3me-2’-dC were shown to stimulate oKG decomposition without
undergoing demethylation (27). In the absence of nucleotide, but the presence of aKG
and 02, AlkB hydroxylates its own Trp 178 (located near the active site, see Fig. 4.2) to
generate a hydroxy-tryptophan side chain. A ligand—to-metal charge-transfer transition
between this modified residue and the oxidized metal site results in a blue protein that
absorbs maximally at 595 nm (28).

This chapter focuses on an AlkB homolog of T. brucei and describes the DNA
repair activity observed for this member of the non-heme F e"- and aKG-dependent

hydroxylase family of enzymes in kinetoplastids.

90

4.2 MATERIALS AND METHODS

4.2.1 Gene identification and multiple sequence alignment

The Basic Local Alignment Search Tool (29) was utilized to search the protein-
encoding sequences of the T rypanosoma brucei brucei genome with the protein sequence
of E. coli AlkB as the query, resulting in the identification of a sequence with the NCBI
accession number XP_844196. The sequence of the identified trypanosomal AlkB
homolog (TbABH) was aligned to representative group 1A (30) AlkB sequences of E.
coli (NP_416716), Brucella abortus (ZP__05894130.1), Pseudomonas putida
(AAN69003.1), Pseudomonas syringae (NP_792910.1), and the first three human AlkB
homologs (ABHI, AAH25787.1; ABH2, Q6NS38.1; ABH3, Q96Q83.1) by using Clustal
W (31) for analysis. Further, the TbABH sequence was analyzed to predict the
subcellular location by several online servers including LOCTree (32), PSORTII

(http://psort.hgc.jp/fonn2.html), SubLoc (33), and ESLPred (34).

4. 2.2 Cloning

A 991 -bp DNA fragment containing TbABH was amplified by PCR using
genomic T. b. brucei strain 427 DNA as a template with “forward” (5’-AGGATATA_C_Q
ALQGAAGACC CCGTGC-3’) and “reverse” (5’-GA GCATCCTCGAG TTCGTTAAG
GAACTCAC-3 ’) primers which introduce NcoI and Xhol restriction sites (underlined),
and T aq polymerase master mix kit (Promega) which leaves a single 3 ’ adenine
nucleotide overhang. The PCR product was treated with a PCR clean up kit (Qiagen,

Inc.) and ligated into pGEM-T Easy (Promega) to create pGEM-TbABH. The pGEM-

91

TbABH plasmid was transformed into E. coli DHSa (Invitrogen), isolated from several
transformants, and sequenced (Davis Sequencing). TbABH was excised from the pGEM-
T backbone by N001 and Xhol restriction and ligated into pET28b (Novagen) which had
been cut previously with the same enzymes, creating pET-TbABH and putting the coding
sequence in frame with a sequence encoding a C-terminal 6-histidine tag. This plasmid

was transformed into the expression strain E. coli BL21 (DE3).

4. 2.3 Protein production and purification

E. coli BL21 (DE3) cells containing pET-TbABH encoding TbABH-His,
(hereafter referred to simply as TbABH) were grown at 30 °C in lysogeny broth (LB)
medium supplemented with 100 ug/mL kanamycin while shaking at ~l60 rpm to an
optical density of 0.4 to 0.6 at 600 nm. Cultures were induced to overexpress the desired
gene by addition of isopropyl-B-D-thiogalactopyranoside (IPTG) to 0.1 mM, and grown
for an additional 4 h, then harvested at 4 °C by centrifugation at ~8,000 g for 8 min. The
cell paste was either used immediately for protein purification or stored at -80 °C.

In a typical purification, 3 mL of binding buffer (30 mM imidazole, 10 mM Tris,
150 mM NaCl, pH 7.9) was added per g of cell paste for resuspension. The protease
inhibitor phenylmethylsulfonyl fluoride was added to 0.5 mM, cells were lysed by
sonication (Branson Sonifier, 3 pulses of l min each, 3 W output power, duty cycle 50%,
with cooling on ice), and the cell lysates were ultracentrifuged at 100,000 g for l h.
Soluble cell extracts were loaded onto a Ni-bound nitrilotriacetic acid column (Qiagen)
pre-equilibrated with binding buffer. The column was washed with binding buffer until

the baseline was reestablished, and proteins were released with elution buffer (150 mM

92

imidazole, 10 mM Tris, 150 mM NaCl, pH 7.9). Fractions containing the purified
proteins, as determined by denaturing sodium dodecyl sulfate—polyacrylamide gel
electrophoresis (SDS-PAGE, 12% acrylamide) (35) and Coomassie staining, were pooled
and dialyzed back into binding buffer by using 12-14 kDa molecular weight cutoff
dialysis tubing (Fisher) at 4 °C overnight with stirring. The concentration of protein was
determined by using its calculated molar absorptivity at 280 nm (45,350 M'1 cm”l
according to the ExPasy protein parameters prediction server
(http://ca.expasy.org/tools/protparam.html) (36). The protein was either used

immediately for assays or was stored at 4 °C and discarded after two weeks or if
precipitation developed. A typical purification yielded approximately 3 mg protein per g

of cell paste.

4.2.4 Gel filtration chromatography

To determine the native Size and Oligomeric state of the protein, purified TbABH
was concentrated to 200 uM (7.4 mg/mL) of protomer in an Amicon centrifugal filter unit
with a 10 kDa molecular weight cutoff and was chromatographed on a Superdex®75 size
exclusion column preequilibrated with binding buffer. Retention times were compared to
those of gel filtration standards (BioRad). The collected fractions also were examined by
PAGE using a 3-12% Bis-Tris Blue native gradient gel (Invitrogen) to verify the major

Oligomeric forms of TbABH.

93

4.2.5 Spectroscopy

UV/visible spectra were obtained by using an HP 8453 spectrophotometer
(Hewlett Packard) equipped with a circulating water bath and magnetic stirrer. TbABH
was concentrated in an Amicon centrifugal filter unit with a 10 kDa cutoff to 265 uM of
protomer in binding buffer. Stock solutions of 10 mM F e" and 50 mM aKG were
prepared by adding the dry reagents to vials, subjecting them to repeated cycles of
vacuum and argon, and adding anaerobic water by using a syringe. Protein was added to
a 1-cm path length, 1.5 mL, quartz cuvette, the sample was made anaerobic by gentle
vacuum/argon cycling, and it was scanned while stirring at 9 °C. Four equivalents of
aKG (to 1 mM final concentration) were added and the sample was re-scanned, followed
by titration with iron in 8 increments up to 500 uM with scanning after each addition.
Data were corrected for dilution in Excel and plotted as difference curves with the
absorbance from the protein alone set as the baseline. The difference absorbance of the
feature observed at 530 nm (AA) was plotted versus the concentration of total added iron
(14) to calculate a Kd for the binding of iron to the aKG-bound active Site using equation
1 (37). In this equation, AAmax is the predicted maximal absorbance change, [Ed is the
total enzyme concentration, and n is the number of iron binding sites per protein

molecule.

Eq. 1 AA = AAma;((Kd+r.+an.n-(a<d+t.+nraflaunts.m"-5)
2 ”[3]

4. 2. 6 Electrophoretic mobility shift assays (EMSA)
Gel band Shiit assays were conducted after incubating 50 ng of a plasmid ds-DNA

substrate (7.5 nM in assay) with increasing amounts of TbABH (up to 20 uM) for 1-2 h at

94

30 °C. This maximum amount of TbABH equates to a final ratio of protein to DNA base
pairs of 1:2. These assays were analyzed by electrophoresis using a 0.8% agarose gel and
visualized via staining with ethidium bromide. DNA binding was evidenced by a shift in
the electrophoretic mobility of the DNA band in the gel upon increasing protein
concentration.

Other gel band shift assays were conducted by incubating 32P—labeled
oligonucleotides with increasing amounts of TbABH and monitoring the effects on
electrophoretic mobility when using a 4% polyacrylamide gel via autoradiography.

Briefly, oligonucleotides (forward 5’-CGA TCC AGA CTC GAC TAC GCC ATC CGA
TCC-3’ and reverse 5’-GGA TCG GAT GGC CTA CTC CAC TCT GGA TCG-3’) were
treated with methyl methanesulfonate (MMS) to 1.5% in 20 mM cacodylate buffer, pH
7.5, for 5 h at 31 °C then ethanol precipitated to recover the DNA. 50 pmol of the
forward oligo (either untreated or methylated) was treated with 50 pCi of 7-3 2P-ATP and
10 units of T4 polynucleotide kinase (Gibco) in the provided buffer for 50 min at 37 °C.
DNA was denatured and purified on a 12% agarose gel containing 7 M urea. Gel slices
containing the labeled DNA oligonucleotides were excised and the DNA extracted,
ethanol precipitated to purify, and quantified by scintillation counting. 50 nM stocks
were made for each of the DNA substrates used in the binding assays, each at 10
kcpm/uL. For ds-substrate, forward and reverse oligos were combined and incubated at
70 °C for 1 min, and then slow cooled to 27 °C to allow hybridization.

For binding assays, 10 nM oligo was combined with increasing TbABH
concentration from 0-30 uM in 10 11L (up to a 100-fold excess of protein subunit per base

pair) containing 20% glycerol as a molecular crowding agent for l h at room temperature.

95

Samples were electrOphoresed on a 4% polyacrylamide gel containing a 37.5:1 ratio of
acrylamide/bisacrylamide in Tris-glycine in a BRL V16-2 system at 20 mA per gel until
the dye front was about half way through the gel. Gels were fixed in a solution
containing 10% each of methanol and acetic acid and dried by vacuum. Dried gels were
exposed to a phosphorescence screen for at least 2 h on a Typhoon 9200 variable mode
imager. The images were analyzed by using Image Quant, with the count values of the
Shifted bands normalized to the total counts in each lane, converted to nM shifted DNA,

and plotted against the total TbABH concentration in the sample. The resulting data were

fit in Sigma Plot to equation 2 for hyperbolic single-site saturation, where Bmax is the

theoretical maximum DNA bound, and used to estimate the Kd of DNA binding. The

results shown are the average of duplicate experiments.

Eq' 2 y : Ernaxigl
Kd+lEl

To test binding of TbABH to another form of damaged DNA, oligonucleotides
containing abasic sites were created and used in analogous EMSA studies. Briefly,
oligonucleotides (forward 5’-AGT AGA CAG CTA CCA TGC CTG CAC GAA GUT
AGC AAT TCG TAA TCA TGG TCA TAG CTA GTA -3’ and reverse 5’- TAC TAG
CTA TGA CCA TGA TTA CGA ATT GCT AGU TTC GT G CAG GCA TGG TAG
CTG TCT ACT-3 ’) each containing a single deoxyuridine (in bold) were hybridized by
heating to 95 °C for 1 min then allowed to Slow cool to room temperature. Theds-DNA
was treated with uracil-DNA glycosylase (N ew England Biolabs) following the

manufacturer’s instructions to induce formation of abasic sites. For the assays, 1 uM of

96

treated or untreated oligonucleotides was incubated with increasing amounts of TbABH
(0-30 uM) in 25 mM Tris, pH 8, containing 50 mM KCl for 20 min at 37 °C. Samples
were loaded onto a 6% polyacrylamide gel (19:1 acrylamidezbisacrylamide ratio) and
electrOphoresed at 90 V in TBE buffer (89 mM each of Tris and borate, 1 mM EDTA, pH

8) and visualized by staining with ethidium bromide.

4. 2. 7 In vitro enzyme assays

Production of formaldehyde fiom methylated DNA was monitored by using a
formaldehyde dehydrogenase-coupled assay which converts the product into formate
under conditions that are essentially irreversible (42). The reaction was monitored
spectrophotometrically at 340 nm to follow the coupled reduction of NAD+ to NADH.
Assays containing 1 uM TbABH, 100 uM aKG, 5 uM Fe", 13 uM methylated 25-mer
poly-dA oligonucleotide (~30 uM methylated bases assuming a typical ~10%
methylation efficiency), 1 mM NAD+, and 0.05 units of formaldehyde dehydrogenase in
50 mM Hepes, pH 8, were mixed in a 300 uL quartz, black walled cuvette and
continuously monitored at 340 nm in a Shimadzu spectrophotometer. A series of control
experiments were carried out, omitting individual components of the TbABH assay. A
standard curve demonstrated that formaldehyde was detectable in a linear fashion from
150 uM down to 5 uM in the assay volume.

Consumption of aKG was monitored by using ortho-phenylenediamine (OPDA)
which is known to react with several a-keto acids (43). Assays containing 1 uM TbABH,
13 uM methylated poly-dA oligonucleotide, 5 11M Fe" , and 100 11M aKG in 50 mM

Hepes, pH 8, were incubated at 37 °C. Samples (225 uL) were taken at selected time

97

points and quenched by addition of 75 uL OPDA solution (10 mg/mL OPDA in 1 M
phosphoric acid adjusted to pH 2 with .25% B—mercaptoethanol). Quenched samples
were heated in a 100 °C block for 3 min, cooled to room temperature, and monitored at
340 nm in a quartz, black-walled, microcuvette by using a Shimadzu spectrophotometer.

The standard curve for the OPDA reaction with aKG was linear from 5 uM to 100 uM.

4. 2. 8 Complementation assays

TbABH was cloned out of pET-TbABH by using PCR amplification primers
(forward 5’-CGTGGAATTC ATGGAAGACC CCGTGC-3’ and reverse 5’-GCT
CCAAGCTTTC ATTCGTTAAG GAACTCAC-3’) that introduced EcoRI and Hindlll
restriction sites (underlined). The DNA containing the gene was excised using those
enzymes and ligated into pUC18 to create pUC-TbABH. This construct was transformed
into the BW25113AalkB cell line (38) and grown to exponential phase before plating 100
uL on LB agar containing 200 ug/mL ampicillin. After allowing the plates to dry, sterile
paper discs soaked in 1% or 5% MMS were placed on top of the agar, the plates were
incubated overnight at 37 °C, and the resulting zones of inhibition were analyzed to
monitor cell stress according to the Kirby-Bauer disc diffusion method (39).

In an attempt to increase TbABH protein production during the complementation
studies, TbABH was cloned into the IPTG-inducible vector pEXT20 (40) by using pET-

TbABH as a template and PCR amplification primers (forward 5’-GAT GAATTCTQ
-ATATAC ATGGAAGACC CCGTGCG—B’ and the same reverse primer as

above) that introduced an EcoRI restriction site (underlined) while preserving the

98

ribosomal binding Site (boxed). The construct was transformed into BW251 l3AalkB, and
the culture was grown, plated, and stressed with MMS as above.

Another vector used to enhance TbABH protein production during
complementation was pMMB67, which was created by and obtained from Dr. Michael
Bagdasarian. This is a low copy number plasmid with an IPTG—inducible T aq promoter,
ampicillin resistance gene, RSF 1010 replicon, and a multiple cloning Site derived from
pUC18. TbABH was subcloned from pUC—TbABH by using the restriction enzymes
EcoRI and HindIII and ligated into Similarly digested pMMB67, and the xenobiotic stress
assays were repeated with BW25113AalkB cultures containing this recombinant plasmid.

TbABH was found to be out of frame in pUC—TbABH, so this plasmid was
mutagenized by using PCR amplification primers (forward 5’-GATTACQ_AA_T
EOE—gems ACCCCG—3’and reverse 5’-CGGGGTCTTC @jCGAATTc
GTAATC-3’) that inserted a base downstream of the EcoRI site (underlined) and before
the TbABH start site (boxed), placing the gene in-frame with the lacZa start Site which
added 7 extra residues to the N-terminal end of the produced protein. The new construct,
denoted pUC-TbABHZ, was verified by sequencing (Davis Sequencing) and transformed
into BW25113AalkB cells. Cultures were grown to exponential phase before being
induced with 50 uM IPTG. After one doubling time of additional growth to allow protein
production, 100 uL of this culture (and the necessary control cultures) were plated on LB
agar containing 200 ug/mL ampicillin and 50 uM IPTG. After reaching dryness, the
Kirby-Bauer disc diffusion assays were repeated using 1% and 5% MMS.

Finally, TbABH was cloned into pWKS30 (41) by using pET-TbABH as a

 

template and PCR amplification primers (forward 5’-CGTGGGTACC EAAGGWTA

 

99

TACCATGG-3’and the same reverse primer used to clone the gene into pUC18 from
pET—TbABH) that introduced a KpnI restriction site, underlined, while preserving the
ribosomal binding Site (boxed) and the start Site of TbABH (bold). The gene was ligated
into pGEM-T Easy (Promega), verified by sequencing (Davis Sequencing), subcloned
into pWKS30 by using the KpnI and HindIII restriction sites, and the latter plasmid was
transformed into the knockout cell line. Kirby-Bauer assays were repeated with this
construct as before.

In order to verify the sensitivity differences between the wild type and alkB
knockout BW25113 cell lines to MMS, a viable cell count assay was performed. Briefly,
cultures were grown to exponential phase in LB medium, stressed with 0.5 % MMS, and
samples were taken at various time points, diluted, and plated on LB agar. Plates were
incubated overnight at 37 °C and the colony forming units (CFUS) were counted. CFUS
were corrected for dilution and plotted as total viable cell count versus time under the

stress condition.

100

4.3 RESULTS AND DISCUSSION

4.3.1 Analysis of the T bABH Sequence

The genome of T. brucei brucei encodes a full length ortholog of the E. coli AlkB
protein, that we have termed T. brucei AlkB homolog (TbABH). TbABH was predicted
to be a nuclear protein by the ESLPred, SubLoc, and PSORTII online prediction servers,
and further identified as a DNA-binding protein by LocTree. TbABH is 23% identical to
E. coli AlkB with the most conserved regions predicted to be located in the double-
stranded B-helix and including all the putative metal-coordinating facial triad residues
(His 213, Asp 215, and His 269) as well as the probable aKG-stabilizing Arg 305 (Fig.
4.3). These residues, along with several others, were completely conserved among the
bacterial and eukaryotic AlkB-related sequences aligned. This is not surprising since the
core fold is one of the only defining features of this enzyme family. In fact, most of the
conserved residues mapped to the B-strands making up the catalytic core of the E. coli
AlkB crystal structure (21). In addition, several more residues were completely
conserved between TbABH and the bacterial sequences including a few components of
the nucleotide recognition lid in E. coli (residues 45-90) known to contact the nucleotide
substrate (Trp 151, Tyr 158, and Ser 161 in TbABH).

Interestingly, there was much greater similarity between the TbABH sequence
and that of the bacterial AlkB sequences than between TbABH and the other eukaryotic
AlkB homologs represented in the multiple sequence alignment. This result is consistent
with this protozoan gene being acquired by horizontal gene transfer. Such is the case for

several other genes in the trypanosomatids including elements of the heme

101

Figure 4.3. Multiple sequence alignment of selected bacterial and eukaryotic AlkB-like
proteins. Residues completely conserved are marked with two dots below the sequence.
Residues conserved among all bacterial sequences and TbABH are shaded black and
indicated with a single dot below the sequence. Residues viewed as being similar based
on the Clustal W alignment software are Shaded grey. The arrows represent the 8 B-
strands that make up the core fold of E. coli AlkB (21). The metal-coordinating triad and

the aKG-stabilizing arginine are marked with asterisks below the sequence. Numbering

   
    
 
  
 

 

   
   
    

is according to TbABH.
100 110 120 130 140 150 160

...l....l....]....l....|....)....I....|....|....l....I....|....|.---... ..
E.Coli_AlkB YTMSVAMTN G ------------------------------------- HL HR P--IDPQTN
B.abortus_AlkB YRMSVAMTNCG ------------------------------------- PV DRT P--ADPETG
P.putida_AlkB LRMAVALTNCG ------------------------------------- T SDQH Pe-SDPVSG
P.5yringae AlkB LSMSAALSSCG ------------------------------------- PL ITDRH --VDPQTG
T.brucei_ABH EVFPNFLTDAEQQEFCRAALLEYGDSRRHPNHLSTHASEPRETTRYEPPM TVGF ---SKSYCK
H.5apien ABHl CNLDKHMSKEETQDLWEQSKEFLRYKEATKRRPRSLLEK ---------- L GY ~--SKKYSA
H.5apien ABH2 ELEKEVEYFTGALARVQVFGK --------------------------------- SVPRKQATYGDAGLTYTF
H.Sapien ABH3 QLCQDVPWKQRTGIREDIT ------------------------------------ YQQPRLTAWYGE--LPYTY

O O I O O

200 210
....l....|....-|.... ..
3. C01 i__A1kB ACLI YA K‘LS .:_.'1v
B.abortus_AlkB ACLI YE R-LS Hg
P.putida_AlkB ACL YL R-LS wg
P.5yringae AlkB ACLI YI -MS w;
T.brucei_ABH SI PP -I “NJ
H.5apien ABHl CGFED—FRAEAGI YRLDST-LGI" .
H.8apien ABH2 SGLTLS P-WIPVLERIRDHVSGVTGQTFNFVLI YKDGCDHIGa'
H.sapien ABH3 SRITM PHWHPVLRTLKNRIEENTGHTFNSLL LYRNEKDSVD g
==i>
280 290 300

. .... .... ... . .. .. ....[....l....l. .l. ..l....l...
E.coli#A1kB ~;T “ IQPLKAGFHP ------------------- LTID
B.abortus_AlkB ‘1' ' ILPLKSGEHE ------------------- RLGP
P.putida_A1kB «fi' “. PIKPGVHP ——————————————————— RLGE
P.5yringae AlkB ifl‘fi ILPIKQABHP ------------------- LLGE
T.brucei_ABH “ PRIMDDCPQYLCPQNETEEEEGEFYWREQMRH
H.sapien ABHl VPRVLPNPEGEGLPHCLEAPLPAVL(l9)YLKT
H. sapi en ABH2 P‘I‘NTHWYHSLPVRK ---------------- KVLA
H. sapi en ABH3 ——————— RKKPPPEEAI’YTYVERVKI PL LLIMEGATQADWQHRVPKEY -------- HSRE

 

O O O O 0.. O O O *0

102

biosynthetic pathway (44), phospholipase A1 (45) and isopropyl alcohol dehydrogenase
(46). Despite TbABH’S greater similarity to the bacterial AlkB-like proteins than to
homologs in eukaryotes, it is distinct in possessing a much longer amino terminus and
inserts of 37, 1, and 19 residues compared to the E. coli protein. The largest insert
extends from within the nucleotide-binding lid of AlkB, the extra amino acid at residue
193 corresponds to an unstructured region on the back face of the E. coli protein in the
view of Figure 4.2, and the 19 amino acid insertion (residues 281 ~299) corresponds to an

unstructured region at the very top of AlkB in the view shown.

4.3.2 General biochemical properties of TbABH

To characterize this protein, TbABH was cloned from T. b. brucei genomic DNA,
expressed recombinantly in E. coli, and the protein purified by affinity chromatography
to about 90% purity as visualized via SDS-PAGE and Coomassie staining (data not
Shown). Figure 4.4 shows that multiple forms of the protein were present as purified
from E. coli. The predominant peak in the chromatogram (panel A) chromatographed as
a monomer while the less prominent peak eluted as a dimer; however, native gel
electrophoresis of the fractions was most consistent with both protein Species forming an
equilibrium mixture of 37 kDa monomers, 74 kDa dimers, and 148 kDa tetramers.

To confirm prOper folding of TbABH, the purified protein was examined for its
ability to form a characteristic chromophore seen in other members of the F ell/aKG-
dependent dioxygenases and associated with binding of the iron cofactor and the aKG
cosubstrate (47). The protein was monitored by UV-visible spectroscopy under

anaerobic conditions, using low temperature to maintain stability, while titrating in OLKG

103

Figure 4.4. Native size and Oligomeric state determination of TbABH. A.
Chromatogram of TbABH analyzed by using a Superdex®75 column and compared with
BioRad gel filtration standards (retention times of y-globulin, 158 kDa; ovalbumin, 44
kDa; and myoglobin, 17 kDa, are shown as inverted triangles above the chromatogram).
B. Native polyacrylamide gradient gel Showing the Oligomeric states and native sizes of
TbABH. The bar on the chromatogram indicates the fractions shown on the gel.

A. V V V

0.20 1

1

0.151

° 1

a 0.10 1

< 0.05.?

0.00 : ¢ ;

LL j; 4 L r l L :L 4 L . _. 4 fiL _. J i 1

4o 45 50 - 55 so 65 70 75
Retention Time (mm)

 

 

 

 

' . .‘H " ‘
' ‘ ‘ . _. v r c > _
rf‘Jr—‘g, -,. . ...“ . . .

. 21'

,r-G'rw, I.

1“ 3* 10" Li). 4" ‘1 "‘
r- ticmfi‘fir‘: Atari .G‘E'i-{r’o ti... ,3;

. «Jens-"rfinzvth‘ir:

.k-ll

1 ‘T' '4 .. A“ .. ‘_ ' . ' , v
. r _ . . . ' 31' ‘ < -
. “3'57, 1“ _. . . ' ' -* l ‘ ‘
r e ' ‘ . V
. . , r .t. . , 7' " ‘ ',.- 5 r r‘ ’r _ . ‘ -
w . ‘ '( m‘r ,~' . v J‘rrzx .fi ~ .
4.1.‘A’Krf-AJIPIW. . Wigwm ,) I U.” 3.; 1,. ., , p. :r .. ...
~ ‘ . r, y . ,
. ' .. 1. ‘ », j" y.
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r
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<9
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r-
w.
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A. v. ‘1
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4
V'.
. v
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104

and Fe". AS shown in Figure 4.5, anaerobic TbABH generated the diagnostic metal-to-
ligand charge-transfer transition at 530 run when both metal and cofactor were present.
The extinction coefficient for this feature was approximately 190 M’1 cm", consistent
with values previously described for members of this enzyme family (37,48). The
relative absorbance change at 530 nm was plotted versus the total concentration of iron
titrated into the protein sample and fit using equation 1. This allowed for calculation of

an Fe'I Kd of ~4 11M with 1.3 i 0.07 iron atoms binding per active Site.

4.3.3 DNA binding by T bABH

To test the hypothesis that TbABH is a DNA repair protein, the ability of this
protein to bind to potential DNA substrates was tested. TbABH was able to bind to
supercoiled dS-plasmid DNA as evidenced by a shift in the electrophoretic mobility of the
DNA band in an agarose gel (Fig. 4.6 A). The development of multiple TbABH-DNA
complexes confounded efforts to quantify the thermodynamics of this interaction.
Therefore, to obtain more quantitative binding data, EMSA assays were performed using
3‘ZP-labeled oligonucleotides that had been left untreated or had been subjected to
methylation by MMS. While TbABH shifted all polynucleotides to some extent when
tested by this method, a clear preference was observed for ds-DNA and especially for
methylated dS-DNA over other compounds tested (Fi g 4.6 B). These band intensities
were analyzed, normalized to the total 32P counts in each lane, and converted to nM DNA
bound. These values were plotted against the concentration of added TbABH and the
resulting curve was fit to a hyperbolic single Site binding equation (equation 2). This

allowed the estimation of a K; of 7.1 i 1.9 uM (Fig 4.6 C). Although these assays

105

Figure 4.5. Spectroscopic evidence for binding of Fe‘1 and aKG by TbABH. A. The
anaerobic UV/visible spectrum of TbABH (266 uM protomer in binding buffer) was
examined for the sample as isolated (baseline), after adding 1 mM aKG, and while
titrating in F ell with stirring at 9 °C. The (Fen-aKG-protein minus protein) difference
spectra shown correspond to the addition of 0, 63 uM, 125 uM, 188 uM, 250 pM, 313
uM, 375 pM, 438 uM, and 500 uM metal ions. B. The intensity of the absorbance
difference at 530 nm was examined as a function of added Fe". The data were fit to

equation 1.

0.1 -

9’

0.08 1

0.06 ~

0.04 r

0.02 J

A Absorbance

 

 

 

 

 

3' 0.07 '1
0.06 a ,_/’"

0.05 5 ./

A Absorbance

 

 

fil T l I

l
o 100 200 300 400 500
Iron (11M)

106

Figure 4.6 EMSA studies of TbABH and various DNA substrates. A. Agarose gel
depicting the shift in mobility of supercoiled pGEXS plasmid (7.5 nM) with increasing
TbABH concentration (shown by the wedge, 0 to 20 nM). B. Native polyacrylamide gel
Showing the Shift in mobility of 32P-labeled ds-oligonucleotide (7.5 nM; untreated on left
and methylated on right) with increasing TbABH concentration (0 to 30 nM). C. Fit of

data from B to equation 2.

 

C. Untreated Methylated

 

 

r ' '

10 i5 i0 25 30
TbABH (pM)

O
(”I

107

contained 10 nM DNA, the process used for methylating the substrate typically exhibits
poor efficiency (~10%); thus, the observation that saturation (Bmax) corresponded to 0.79

:1: 0.08 nM DNA indicates that nearly stoichiometric binding (close to 80%) of the
methylated substrate was bound. In contrast to the above results Showing TbABH’s
preference for binding to methylated ds-DNA, other oligonucleotides were bound with
lower affinity. Very little binding was observed for ss-DNA with no differences noted
between the untreated and the methylated substances (data not Shown). Furthermore,
TbABH did not exhibit enhanced binding to DNA containing an abasic site; where as this

binding has been noted for human ABHl (49).

4. 3.4 Examination of DNA repair by TbABH

In an attempt to obtain direct evidence for the demethylation reaction expected of
a functional AlkB, two in vitro assays were examined. The purified recombinant protein
was used in a formaldehyde dehydrogenase-coupled reaction that allows for detection of
released formaldehyde by monitoring an increase in absorbance at 340 nm due to the
conversion of NAD+ to NADH as part of the coupled reaction. While there was an
indication of turnover in one experiment using this assay, only background rates were
found in follow-up studies where the protein-free control also yielded a slightly positive
reaction. Efforts to demonstrate OLKG consumption by an OPDA assay also were
inconclusive due to the low sensitivity of measuring substrate loss.

As an alternative approach, TbABH was tested for the ability to complement an
alkB knockout in an E. coli cell line. The alkB gene of BW251 13AalkB cells is replaced

with a kanamycin resistance cassette resulting in 3.5-fold greater susceptibility to

108

methylation damage by MMS as evidenced by the decreased viable cell counts over time
when exposed to 0.5% of this methylating agent (Fig. 4.7). The alkB knockout cell line
was transformed with pUC-TbABHZ (which placed the trypanosomal gene in the correct
reading frame while adding 7 extra residues to the N-terminus of the protein) and tested
for MMS sensitivity by the Kirby-Bauer method. At 1% MMS, wild type cells Showed
no inhibition of growth while the alkB knockout exhibited an average zone of inhibition
(the distance between the edge of the paper disc and the edge of non-growth) of 4.0 i 0.7
mm. The complemented strain containing pUC-TbABH2 partially complemented the
knockout exhibiting a zone of inhibition of 1.5 i 0.3 mm (Fig. 4.8).

To extend the alkB complementation approach, the trypanosomal gene was cloned
into several other vectors and the disc diffusion assays were repeated. The BW25113 cell
line does not contain the DE3 lysogen and thus cannot support T7 dependent expression
plasmids, So efforts focused on three alternative vectors which would use the natural
methionine start of TbABH. However derivatives of pEXT20, pMMB67, and pWKS30
containing TbABH failed to produce soluble protein in this cell line so the

complementation studies could not be performed.

109

Figure 4.7 Time course viability test with wild-type and AalkB BW251 13 cell
lines stressed with 0.5% MMS. BW25113 wild type (filled symbols) or BW25113AalkB
(open symbols) cultures were grown to exponential phase, stressed with 0.5% MMS,
sampled in a time course, diluted and plated to determine viable cell count. The time of
exposure to MMS required to reduce viability by half was 3.5 fold greater for the wild

type than the knockout cultures.

1.E+09

1.E+08 I

1.9071 ‘
...l 1.E+06 -
é 1.E+05 J
a 1.E+04 4 I
U 1.E+03 i

1.E+02 . T I

1.E+01 l r

1.E+00 ‘Wfir—Q—m—Q

010 20 30 40 50 60 70 80 90100110120

Time of exposure (min)

 

110

Figure 4.8 Complementation of an E. coli alkB mutant with T bABH under alkylation
stress. BW25113 cells containing either pUC18 or the derivative pUC-TbABH2 were
stressed with 1% MMS according to the Kirby-Bauer method (39). Plates were incubated
overnight at 37 °C and the resulting zones of inhibition were measured. Below is a
representative experiment showing the wild type cells containing pUC18 (labeled WT),
AalkB cells containing pUC18 (AalkB), and AalkB cells containing pUC-TbABH2

(TbABH). The zones of inhibition are shown by the black lines and white dashed circles.

WT AalkB TbA BH

 

lll

4.3.5 Conclusions

Trypanosomes contain a full length ortholog of the E. coli AlkB which exhibits
characteristics representative of the Fe" and aKG dependent dioxygenase superfamily of
enzymes including the formation of a diagnostic metal-to-ligand charge-transfer
chromophore when incubated with the metal and cofactor under anaerobic conditions.
Further, while E. coli AlkB binds DNA only very weakly (50), TbABH forms a tight
complex and exhibits a clear preference for alkylated ds-DNA. Although direct evidence
for DNA repair was not obtained in vitro, an E. coli cell line deficient in alkB was
complemented by the expression of TbABH upon alkylation stress conditions confirming

its assignment as an AlkB protein.

112

10.

11.

REFERENCES

Barrett, MP, Burchmore, RJ, Stich, A, Lazzari, JO, Frasch, AC, Cazzulo, JJ,
Krishna, S. 2003. The trypanosomiases. Lancet 362: 1469-1480

Pace, NR. 2009. Mapping the tree of life: progress and prospects. Microbial Mal
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117

CHAPTER 5

PERSPECTIVES

118

5.1 INTRODUCTION

Enzymes of the non-heme iron and a-ketoglutarate (dKG) dependent dioxygenase
superfamily are ubiquitous in nature and versatile in catalytic activity. Members have
been identified in nearly every branch of life and catalyze oxidative reactions ranging
from hydroxylations to desaturations, ring formation, or ring expansion to name a few
(1). The defining features of this family are the double-stranded B-helix core fold and the
requirement for a ferrous ion at the active site. Most members oxidatively decarboxylate
aKG to form C02 and succinate while creating a highly reactive ferryl-oxo species that
then carries out the primary oxidative reaction (1). One branch of phylogeny for which
no direct activity for this enzyme family has been Shown is the kinetoplastid flagellates
including trypanosomes.

Trypanosomes are the source of medical and economic challenges in their
endemic areas, causing disease in humans and livestock (2). These parasites have unique
anatomy and physiology (see Chapter 1) and complex relationships with their
mammalian and insect hosts. They undergo drastic physiological changes during their
life cycle and persist in mammalian infection through host immune system evasion
involving a long studied, yet poorly understood, process called antigenic variation (3).
For all of these reasons, trypanosomes are interesting topics of study. The experiments
described in this thesis sought to identify and characterize several putative F e" and aKG-
dependent hydroxylases from trypanosomes in order to increase understanding of these

parasites and expand knowledge of this remarkable enzyme family.

119

5.2 SUMMARY OF RESULTS

5.2.1 T LP5 and TLP7 are unlikely to participate in pyrimidine salvage

Two T rypanosoma brucei genes were identified based on sequence similarity to
thymine 7-hydroxylase from Rhodatorula glutinis and termed T LP5 and T LP7 . The
genes were cloned into Escherichia coli and the recombinant proteins expressed, purified
by column chromatography, and characterized. Both proteins were soluble as expressed
and existed predominantly as monomers in solution. In addition, proper folding and
binding of the Fe" and aKG cosubstrates were verified by observation of a characteristic
metal-to-ligand charge-transfer (MLCT) feature by UV-visible spectroscopy. However,
neither protein bound to the tested nucleic acids or exhibited enzymatic activity as
measured by oxygen consumption or succinate production.

Steady state mRNA levels of TLP5 and TLP7 were tested by quantitative reverse
transcriptase PCR and were found to be several fold higher in the bloodstream form (BF)
of the parasite over the procyclic form (PF) for both genes, suggesting that the RNAs are
stabilized in this life stage. Epitope tagging of the chromosomal copy of TLP5 was
attempted in order to be able to test the subcellular localization and expression levels of
the protein, but this effort failed on six separate attempts.

Protein production was verified in trypanosome cell extracts by western blotting
with a polyclonal antibody raised against TLP5. The antibodies were Shown to be cross
reactive against TLP7, and both proteins were identified in extracts of both BF and PF
parasites. Each cell form also contained a cross reactive band of higher molecular weight

suggesting possible posttranslational modification of these proteins in the trypanosome

120

cell. Pull-down assays were performed to identify possible protein partners of TLP5 and
TLP7 in the cell by passing trypanosome cell extracts over column-bound recombinant
TLP5 or TLP7 and examining the eluant on a polyacrylamide gel, but no interacting
proteins were observed.

In efforts to detect enzymatic activity from these proteins in their natural states,
cell extracts were incubated with possible substrates along with cosubstrates and
analyzed for product formation by mass spectrometry, but no product formation was
observed. In addition, a homology model of TLP5 was created and used in docking
studies in an effort to generate hypotheses about the enzyme specificity; although several
heterocyclic ring structures that resembled nucleotides were identified as the best ligands,

no convincing potential substrates were identified.

5.2.2 JBP1 is inactive in its recombinant farm

Trypanosomes contain a unique base in their nuclear DNA, B-D-
glucosylhydroxymethyldeoxyuracil or base J, which replaces up to 1% of thymine bases
(4). Extensive evidence has been obtained that JBP1, a protein Shown to bind specifically
to J -containing DNA, is responsible along with JBP2 for the introduction of base I into
chromatin in viva, though no in vitro activity has been reported (5,6). Recombinant
J BP1 , purified from E. coli, was shown to fold properly and bind Fe“ and uKG via
observation of the MLCT feature; however, no activity was observed when it was tested
for in vitro activity by monitoring succinate production via high performance liquid
chromatography or oxygen consumption via oxygen electrode. While it has been

established that JBP1 and J BP2 are critical to the production of base J in trypanosome

121

DNA, the in vitro systems tested were insufficient to observe any activity of these

enzymes.

5.2.3 T bABH is a DNA-binding protein homologous to E. coli AlkB

TbABH is an ortholog of E. coli alkB, and was predicted to be a nuclear, DNA-
binding protein. Its peptide sequence of the catalytic core aligned well with bacterial
AlkBs of group 1A (7) including all metal-coordinating residues and several other highly
conserved amino acids known to make contact with the nucleic acid substrate in E. coli
AlkB (8). TbABH was cloned into E. coli and the protein expressed and purified.
TbABH was found to be soluble and in equilibrium between monomer, dimer, and
tetramer forms. Proper folding was verified by the formation of the MLCT feature when

anaerobic sample was incubated with F e" and orKG, and titration studies provided a
ferrous ion Kd of approximately 4 pM.

TbABH bound to supercoiled plasmid DNA, as evidenced by electrophoretic
mobility shift (EMSA) assays in agarose gels, and to linear alkylated oligonucleotides, as

evidenced by EMSA assays in polyacrylamide gels. The latter EMSAS, performed with
varying amounts of TbABH, allowed determination of a methylated DNA Kd of 7 pM

with near stoichiometric binding to the alkylated portion of the substrate, given the
typical low alkylation efficiency. The specific shift observed with methylated DNA was
not observed with untreated DNA, and was not due to the creation of abasic Sites
indicating that TbABH binds preferentially to methylation-damaged DNA.

TbABH was tested for in vitro activity by monitoring oxygen consumption via

oxygen electrode, aKG consumption via the OPDA assay, and formaldehyde production

122

via the FDH-coupled assay. Unfortunately, none of the approaches provided conclusive
results. TbABH was also tested for the ability to complement an alkB knockout in an E.
coli cell line stressed with methylmethane sulfonate. The complemented cell line
containing pUC-TbABHZ was approximately two fold more resistant to MMS growth
inhibition than the knockout cell line containing underivatized pUC18. This
complementation along with the DNA binding confirms the assignment of TbABH as a
functional AlkB-like protein in T. brucei expanding the knowledge of DNA repair

mechanisms in this organism.

123

5.3 CONCLUSIONS AND FUTURE DIRECTIONS

Trypanosomes contain several representatives of the F e" and aKG-dependent
dioxygenase superfamily, and while all the proteins investigated in this study were
expressed well in E. coli and yielded soluble protein that was properly folded and capable
of binding iron and aKG, none of the in vitro assays detected enzymatic activity. It may
be that trypanosomes contain some stabilizing or activating factor that is missing in our
assay conditions. Also, it is possible that these proteins require some type of
posttranslational modification for activity, consistent with the results obtained in western
blots of trypanosome cell extracts with anti-TLP5 antibodies where prominent cross-
reactive bands were observed. On the other hand, I was unable to detect thymine 7-
hydroxylase activity using a mass Spectrometry assay after incubating thymine with
trypanosome cell extracts, perhaps because the protein levels were too low to allow
detectable product formation or the substrates tested may have been incorrect.

In order to address these problems, one could attempt to purify the proteins
directly from trypanosomes, bypassing the recombinant system entirely, so as to maintain
the native form of the protein as translated and modified in the cell. This would pose a
new problem of yield, however, Since trypanosomes do not grow to a high density and
the proteins of interest would not be over expressed. Many culture flasks would be
needed to obtain the number of cells required to achieve a comparable amount of protein
purified from 1 L of an E. coli culture.

Another approach would be to use trypanosome cell extracts as an additive in in

vitro assays with recombinant protein. This could possibly provide a needed stabilizing

124

or activating factor, however then the reactions become less defined and any observed
activity cannot be directly attributed to the recombinant protein. It would provide a
starting point though, and the in vitro assay could be refined and optimized once it was
known that something in the cell extract could render the protein active.

Finally, one could look into other potential enzyme family members in these
amazing parasites. The genome of T rypanasama brucei contains many orthologs of F e"
and aKG-dependent dioxygenases and studying several of these could lead to further
understanding of trypanosome biology and new therapeutic targets. As one example,
locus Tb09.211.3730 (XP_827514) encodes an ortholog of the oxygen-sensing
asparaginyl hydroxylase (FIH) of humans (25% identity over 343 amino acids). Whereas
the human enzyme is 349 amino acids, the trypanosomal protein is 1145 amino acids in
length and consistent with a multi-domain architecture. The role of FIH in humans is
oxygen-sensing via hydroxylation of the hypoxia inducible factor (HIF) (9), so it is
reasonable to suspect a similar function of the protozoal protein. In this regard, the
bloodstream form of the parasite strictly uses glycolysis for its energy needs while the
intra-insect or procyclic form uses oxidative phosphorylation; thus, oxygen sensing plays
a critical role in protozoan cellular regulation. It would be interesting to investigate this
trypanosomal F IH homolog and determine if it has asparaginyl hydroxylase activity by
testing several Asn-containing possible substrates by the methods used previously or a
more sensitive approach utilizing a radiolabeled cosubstrate such as 1-[ l4C]-OLKG.

Another example of potentially interesting proteins to study is a group of highly
related sequences arranged in a cluster. Trypanosomes contain large gene arrays within

polycistronic units Spanning megabase regions of their chromosomes (10). Some genes

125

 

whose protein products are needed in abundance, such as tubulin, are known to appear in
groups of tandem copies in order to obtain more of the primary transcript. Therefore, the
presence of five tandemly repeated genes in T. brucei (Tb927.2.6180, 6210, 2130, 6270,
and 6310) that encode nearly identical proteins of 319 amino acid residues and are likely
to be Fell/dKG dioxygenases based on sequence comparisons is interesting because it
implies that the protein product of these genes is important for the cell and needed in
abundance. While there is no close homolog to these sequences to suggest a function for
them, it would be interesting to pursue a screening approach to identify possible
substrates. This could be accomplished using l4C-labeled aKG that would release l4C02
upon its oxidative decomposition. Any positive results from those assays would have to
be followed up with substrate specific assays to confirm its assignment as a substrate.
Trypanosomes are of great interest because they present many health threats to
society and their unique biology offers exceptional challenges to scientists. They will
continue to be studied even after effective and affordable cures of their diseases are
available because they offer an interesting window into phylogenetic relationships
represented in the tree of life. Having branched so early from the eukaryotic lineage,
they are very divergent from the more widely studied model organisms for eukaryotic
systems, such as yeast (11). They harbor several genes that appear to have been obtained
by gene transfer from bacteria and others that seem to be distantly related to plant
sequences based on alignments. The F e"/aKG dioxygenases represent an important
enzyme family responsible for many reactions in the cell including several related oxygen
sensing functions. Sensing oxygen levels is critical to any organism and especially for

trypanosomes as they undergo changes associated with their life cycle which includes

126

forms exhibiting very different oxygen utilization. I am confident that in time, members
of this enzyme family will be found to be involved in the regulation of these processes,

expanding their roles across nature.

127

10.

11.

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