23$ l|H1\NU\lml\l\lWNUlt\\|fi|\\\l||fl\lm|WWWI .THS mess”; HIGH,“ / 572 LIBRARY WWW/W 3 1293 047/” WWW! Mlchigan State 883 Unlversity This is to certify that the thesis entitled EpitOpe mapping and functional analysis of monoclonal antibodies to DnaA protein presented by Wenge Zhang has been accepted towards fulfillment of the requirements for M. 8. degree in Biochemistry Major profess Date 34-46 07639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE N RETURN BOX to remove We checkout from your record. TO AVOID FINES return on or before dete due. DATE DUE DATE DUE DATE DUE MSU le An Affirmative Action/Equal Opportunity lnetituton monur EPITOPE MAPPING AND FUNCTIONAL ANALYSIS OF MONOCLONAL ANTIBODIES TO DnaA PROTEIN By Wenge Zhang A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE Department of Biochemistry 1996 ABSTRACT EPITOPE MAPPING AND FUNCTIONAL ANALYSIS OF MONOCLONAL ANTIBODIES TO DnaA PROTEIN BY Wenge Zhang DnaA protein of E. coli is required for the initiation of DNA replication from the chromosomal replication origin, oriC. In order to correlate the struc- ture of DnaA protein to its functions, monoclonal antibodies to DnaA protein were generated, and their inhibitory effects on the activities of DnaA protein were characterized. The epitopes of these monoclonal antibodies were precisely mapped. Monoclonal antibodies M1, M10, M12, M36, M43, M48, M60, M85, M100 and A3 recognize continuous epitopes located within amino acid residues 86-148 of DnaA protein. M7 and A22 recognize conformational epitopes. The failure of these antibodies to inhibit activities of DNA binding and ATP binding indicates that amino acid residues 86-148 of DnaA protein are not involved in these activities. The epitope bound by monoclonal antibody M7 may be in- volved in interaction with DnaB protein. The inhibitory effect of M1 and M60 suggests a possible interaction between DnaA protein and subunits of DNA polymerase III holoenzyme. To my loving daughter, Julie Ann Ju 6 my husband, Nengjiu Ju and my parents ACKNOWLEDGMENTS First of all, I would like to thank Professor Jon Kaguni for his guidance and patience during the past few years. Without his help, this work would have never been accomplished. Thanks are also due to Professor Laurie Kaguni and my guidance committee members, Professor John Wilson and Professor Zach Burton for their sugges- tions and advice. I wish to express my appreciation to Professor Mel Schindler and Professor John Wang for their very kind help. I would also like to express my thanks to members of the Kaguni group, Kevin Carr, for his generous supply of DnaA protein; Mark Sutton, for his insightful suggestions and providing his dnaA mutants; Carla Margulics, for showing her enthusiasm toward science and many other things, for the memory of our adventurous ski trip, and lots more; David Lewis, for his always kind help. Thanks are also due to Jianjun Wang, Yuxun Wang and Carol Farr. I would like to thank all my Chinese friends, too many of them to be men- tioned here; and Ann Tobin, for the friendship we shared and cherished. Finally I would like to thank my husband for his support, and my parents for everything they gave me. iv Contents LIST OF TABLES vii LIST OF FIGURES viii 1 Literature Review 1 1.1 The initiation of E. coli chromosomal replication ......... 2 1.1.1 The replication origin, oriC ................ 2 1.1.2 The initiation of E. coli chromosomal replication at or‘iC 7 1.2 DnaA protein ............................ 11 1.2.1 dnaA mutants ........................ 11 1.2.2 Biochemical properties of DnaA protein . . ........ 13 1.2.3 Structural and functional domains of DnaA protein . . . 18 1.3 Objective of the thesis ....................... 21 2 Epitope mapping and functional analysis of monoclonal anti- bodies to DnaA protein 28 2.1 Introduction ............................. 28 2.2 Experimental Procedures ...................... 31 2.3 Results ................................ 38 2.3.1 Identification of continuous epitopes ............ 38 2.3.2 A22 and M7 appear to recognize conformational epitopes 42 2.3.3 DNA binding is not inhibited by monoclonal antibodies . 45 2.3.4 ATP binding is not inhibited by monoclonal antibodies . 47 2.3.5 Several monoclonal antibodies inhibit oriC replication and ABC priming ........................ 47 2.3.6 Antibody inhibition of DNA unwinding by DnaA protein 52 2.3.7 Antibodies inhibit the interaction between DnaA and DnaB 54 2.4 Discussion .............................. 60 3 Summary and Perspective 69 List of Tables 2.1 Deduced epitopes of monoclonal antibodies to DnaA protein . . 40 2.2 The influence of monoclonal antibodies on ABC priming . . . . 50 2.3 Summary of the linear epitopes and the inhibitory effects of mon- oclonal antibodies to DnaA protein ................ 59 List of Figures 1.1 1.2 1.3 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 Structural features of the E. coli chromosomal replication origin, oriC ................................. A model for replication initiation at oriC ............. Structural and functional domains of DnaA protein ....... Monoclonal antibody M7 and A22 recognize conformational epi- topes ................................. DNA binding activity is not inhibited by monOclonal antibodies M85 does not inhibit the ATP binding activity of DnaA protein Inhibition of oriC replication by monoclonal antibodies ..... M7, A3, M100 and A22 inhibit F1* formation at 2 mM ATP . . Monoclonal antibody ZB (against hexokinase) does not inhibit the interaction between DnaA and DnaB ............. Monoclonal antibodies M1, M10, M12, M36, M48, M100 and A3 inhibit the interaction between DnaA and DnaB protein Monoclonal antibodies M1, M7, M10, M43 and M85 interfere with the interaction between DnaA and DnaB proteins ..... 5 9 20 44 46 48 51 53 56 57 58 Chapter 1 Literature Review The Escherichia coli (E. coli) chromosome is circular and is composed of 4,720 kilo-base-pairs (kb). E. coli chromosomal DNA replication begins at a unique site, oriC, proceeds bidirectionally, and terminates at terC. The leading strand is synthesized continuously and the lagging strand is synthesized discontinu- ously. Both strands are synthesized in the 5' to 3’ direction. Replication of the chromosome is regulated not only to ensure the precise timing of replication in the cell cycle, but also to ensure that replication occurs only once per cell cy- cle. As in most macromolecular processes, the replication of the chromosome is regulated at initiation of DNA replication. The dnaA gene product is the initia- tor of chromosomal replication. Physiological and genetic studies indicate that DnaA protein plays a positive role in regulating the initiation of chromosomal replication. 2 1.1 The initiation of E. coli chromosomal repli- cation 1.1.1 The replication origin, oriC Genetic studies first indicated that E. coli chromosomal replication initiates at a unique site, termed as 011C, in the vicinity of dnaA-ilv [16]. The oriC' locus was then cloned as a 9 kb EcoRI fragment based on its ability to confer autonomous replication to a DNA fragment which is nonreplicating by itself and contain the gene for l? lactamase [1]. The recombinant DNA containing the 9 kb EcoRI fragment not only confers ampicillin resistance to bacteria harboring . it but also was readily integrated into the E. coli chromosome by homologous recombination. Through mapping the integration site, the location of the origin on the chromosome was determined to be near 83 min of the E. coli genetic map [1]. Subcloning of this fragment located 011°C to a 422 _bp DNA fragment [2, 3]. The minimal sequence of the E. coli replication origin is composed of 245 base pairs. This was determined by selectively deleting flanking regions of a cloned DNA fragment containing oriC and measuring replication function by plasmid maintenance in E. coli [4]. An AT-rich 12-base pair segment near the left boundary of oriC is required for the maintenance of a pUC- oriC plasmid, but can be replaced by AT-rich vector sequences [5]. Therefore, the minimal DNA sequence for E. coli chromosomal replication should extend 12 bp further to the left. Sequence comparison of the replication origin of E. coli with those from five 3 other Enterobacteriaceae species (Fig. 1.1) [15] revealed regions of conservation among 071°C homolog. Highly conserved sequences are separated by regions of variable sequence but of constant length. Analyses of mutations that affect the function of oriC led to the proposal that oriC consists of two essential sequence elements: sites for initiation protein binding, and spacer elements for the precise arrangement of the protein binding sites [21]. There are several prominent features within the conserved regions (Fig. 1.1). First, the four highly conserved 9-base pair repeats TTAT(A/C)CA(A/C)A serve as binding sites for DnaA protein, and are referred to as DnaA boxes (Rlé R4) [6]. The orientation of the DnaA boxes are inverted with respect to each other. DNasel footprinting analysis defined a possible fifth DnaA box with the sequence TCATTCACA [7]. This DnaA box is between an IHF binding site and DnaA box R2. When certain bases in the DnaA boxes are changed, reduced DNA binding is observed [24]. Any base pair insertion between R1 and R2 impairs or‘iC function [25]. Insertion of 10 bp between R2 and R3 [43] or insertion of 2 kb between R3 and R4 in 011°C is tolerated [17]. Second, three highly conserved AT-rich 13-mers are present near the left boundary of oriC’, each starting with GATC. The DNA helix at the 13-mer repeats is thermodynamically unstable in the absence of Mg”, exhibiting hy- persensitivity to the single-stranded DNA specific P1 nuclease in a negatively supercoiled plasmid [10]. In the presence of Mg“, this region remains base paired. However binding of DnaA protein to oriC’ in the presence of Mg2+ leads to the duplex opening at the AT-rich 13-mer region [8]. By contrast, IciA pro- tein binds to the 13-mer repeats to inhibit unwinding by DnaA protein and replication initiation [9] . The left 13-mer can be replaced by different DNA sequences with similar AT content [5, 10], suggesting that AT-richness in this region is sufficient for oriC' to function. The precise sequence of the right 13-mer is required for 011C to function [5, 8]. For the middle 13-mer, there are controversial results regard- ing whether the precise sequence or mere AT-richness is important for 011°C to function [5, 11]. The Dam methylation site, GATC, in which the adenine residue is methy- lated by DNA adenine methylase, is present 11 times within oriC. While only two are expected in the 245 bp origin region based on random distribution statistics, eight out of the eleven methylation sites are conserved. The first four nucleotides in the AT-rich 13-mers are also Dam methylase recognition sequences. In vivo and in vitro experiments showed that Dam methylation of 011C is involved in regulating the timing of initiation. The replicated chromosomal DNA is in a transiently hemi-methylated state by virtue of pairing of the methylated template strands with the newly syn- thesized DNA that is not. The hemi-methylated oriC' appears to be inactive in DNA replication in vivo [12]. Hemi-methylated oriC plasmids cannot transform dam mutant (defective in DNA adenine methylase), but unmethylated and fully methylated plasmids transform at high efficiency [12]. While no significant dif- ference in the binding of unmethylated, hemi-methylated and fully-methylated oriC' to DnaA protein was observed [18], only hemi-methylated oriC has an l bl" E1“ Ball I WI 100 I C I tum-msmcTMAMnMGATcrnrrTArrTAnAGAIcrnnnTANc:GAICTcTTAnAGGATCGnnnnnnnnTeTGGATA/x nngmncnnn ACACCTATTg ..... TEE: T ..E A.. E. G C .T CACTGCCC II CAAGG.TCCCGC ..... EEEC T ..C T.. G. E C .T CBCCAGGC CCCGG.TCCTGT ..... TEAE..T .. ..€ -.. C. C C .T ACTCTCTA CTCGG.TCCACG ..... TEAE..T .. ..¢ -.. . G. C T .T GCTTETCT GTCAG.TCCGCG ..... .A .6A T. . . A. C - A- . TCGTGTTG GTBATTATTCAT ACCTTAACACTA T T.T --A A T- - ' .- A CAGGTTTC CTGGA.AATGAT 10! 1‘50 Am I: _2_ “TWA'GAICAAMM'ITMMOGGATCMNNCIGIGA-'AIGATCGGIEA—TCnTys"C‘GTATANCIGGG—LTCMMTG"; 960TTAI-CACAQCHCAAM TT. T . T .CAACC GCAAA ATTAA. -- . . —.C 56A. C . G . G.G A ACT AA.A . .TCCGT EGAAA. ACTAG. -- ... C. (NET. C . ... .A C GGTAC A ACT AT.T . .ACGCT AAGAA. ACAAT. -- ... C.GTT.C . ... . .C . AG.G 6 OCT ETuT E .CCCTT AAGAA. GCTTT. -- ... C.GGT.C . .A .AAG. C A GCA AT-A . .GACAA EECGTT CTTAC. -' ... C.AGC.A . ... . TT TGG.T A EGA CAAC - TGGATC TCTTT. AAATCT TC T-- TGATCGA G TTC A .A .CGAT. G G (:06 T 10' Hindi l l ____(250 I4 I r MAI-I: IACnnnanzTTchGGATMCTACr-GGTTnnTCc-ncc{TttnAnCA; AnnATCCMAntnGAnnnnnn-GAT : MRSUS 55m c e: .‘T‘A..AACA E .C . C ...EA. CAA.CT.CCTCA E C CTA-.TCECAC'.. 2 “film 0011: : CG A .C . C CA. CAA.CT TCC C .GJ ATE..TCGCA - Schindler Wind-il- ECAT..TCCG A .C . .C A CAA.CT TTE.‘ .EEE EAA..ACCTCCE “film“! W. TTCAEC.AACE A AC . .C ...GA. CAA.CT.TTA.G T C E A.. T- mitlh Wm .ACEC..TCEE G .A C .E C..TTA ACCACAATTA.‘ .T.E TTCA. CG-A. . ' ' E.T AA 5 .A . AA .AAAC. ACT.BA CTT C TT.C EAToTTTTGEGT. T I VIM-£0 myi- M‘Nnol OIIC 0 an an "0 "n a 'o 0 (D 0 k / -—I-‘-7’ w ‘35 .'_. r____1 m IHF mrs R3 R4 pgrd Al-rich W‘bc region 0 lJ-mor ' DnoAbox Figure 1.1: Structural features of the E. coli chromosomal replication origin, 0110 Upper part: Replication origin sequences from six bacterial species are aligned to show sequence conservation. GATC methylation sites are underlined. AT- rich 13-mers are indicated. DnaA boxes are marked as R1, R2, R3, R4. The fifth is not shown. A large capital letter indicates a consensus base in all six species. A small capital letter indicates that the base is conserved in five out of the six sequences. A lower case letter indicates that the base is present in four or three origins. A bold capital letter indicates a base substitution that inactivates the origin [35]. Lower part: A schematic representation of the oriC region and nearby promoter regions. Filled boxes are DnaA boxes. IHF and F18 protein binding sites are shown. The open circles represent 13-mers [47]. increased affinity for the outer membrane [46] . The outer membrane fraction inhibits the replication of hemi-methylated 011°C plasmid specifically. Therefore, a membrane sequestration model has been proposed: Dam methylation regu- lates the timing of initiation by affecting the formation of a complex between hemi-methylated 0110 and the outer membrane [13]. This complex prevents DnaA protein from interacting with 011°C. Initiation can occur again only after remethylation of the new strand, resulting in the dissociation of ariC from the outer membrane to allow DnaA protein to bind and form an initiation compe- tent DnaA- 011°C complex. The isolation of mutants which can transform dam" cell with fully methylated 011°C plasmid led to the identification of the squ gene ° that is required for sequestration of 011°C [22]. Squ protein has also been shown to be responsible for the binding of the membrane fraction to hemi-methylated 0110 [23]. Binding sites for IHF (integration host factor) and F13 (factor for inversion stimulation) are present in 011°C. In viva and in vitra studies showed that IHF protein facilitates the duplex opening induced by DnaA protein whereas FIS is inhibitory [19, 14, 20]. Mutations in the individual DnaA boxes, 13-mers and Dam methylation sites of 011°C abolish its function as an origin for replication [21]. These observations and the conservation of nucleotide sequence of ariC among Enterobacter‘iaceae suggest the existence of a conserved mechanism for the initiation of chromosomal DNA replication. 7 1.1.2 The initiation of E. calz' chromosomal replication at 011°C Genes required for E. cali chromosomal DNA replication were originally identi- fied by genetic studies through the isolation and genetic mapping of conditional lethal mutants to the chromosome. These studies supported subsequent work on the cloning of these genes and overproduction of respective gene products by recombinant DNA methods. Two achievements made it possible to study the replication initiation in vitra, that then led to significant findings on the biochemistry of the initiation process. The first is the cloning of 011°C into a plasmid for use as a substrate for in vitra assays. The second is the develop- ment of a reconstituted system for in vitro replication with purified enzymes [48] . Required proteins for replication were identified biochemically by omitting individual enzyme component from the system, and were divided into three functional groups. Initiation factors including DnaA, HU, DnaB, DnaC, DNA gyrase, and RNA polymerase recognize the 011°C sequence to form an initiation intermediate. Elongation factors (DnaB, primase, SSB, DNA gyrase and DNA polymerase III holoenzyme) serve in DNA chain elongation. Specificity fac- tors (topoisomerase I and RNase H) maintain dependence on ariC—containing plasmids by suppressing initiation of DNA synthesis at other sites [48]. Utilizing the in vitro reconstituted system, the initiation process was studied in detail to identify several successive stages. A speculative model for the ini- tiation of replication from 011°C has been proposed (Fig. 1.2) [30] that includes initial complex, open complex, prepriming complex, priming complex, followed by bidirectional replication. First, DnaA protein binds cooperatively to the DnaA boxes in 011°C, forming the initial complex. Electron microscopy studies suggest that this complex consists of 20-40 DnaA protein monomers wrapped around by the origin DNA [25]. Both ADP-bound and ATP-bound forms of DnaA can bind to linear or supercoiled DNA [8]. However, footprinting analysis revealed that the ATP form of DnaA protein binds more specifically to the DnaA boxes, whereas the nucleotide-free form binds to other regions as well [31] . ATP complexed with DnaA and a supercoiled ariC plasmid are required for subsequent stages [8, 24]. Once 011°C is bound by DnaA protein, the AT-rich 13-mer repeats in 011°C are partially unwound. This duplex unwinding was detected by sensitivity to cleav- age in this region by single-strand-specific P1 nuclease [8]. DnaA protein may interact with the top strand of the 13-mers to induce duplex opening. Primer extension from two primers complementary to the top and bottom strand se- quence outside the 13-mer region was used to measure cleavage of each individ- ual strand by P1 nuclease. The cleavage of the top strand is limited to only part of the middle 13-mer, while the cleavage of the bottom strand spans the three 13—mers and the adjacent AT-rich region [31]. The uncleaved region of the top strand may be due to DnaA protein protection. Mutations that only alter the specific sequence but preserve the AT-richness in the middle and the right 13.mers result in pronounced reduction of 011°C function [57] . This sug- gests a sequence-specific interaction of DnaA protein with the middle and right 13-mer. This open duplex DNA with bound DnaA protein is referred to as the 38. dnaB dnaC ATP P1 INITIAL OPEN COMPLEX COMPLEX PRIMING AND can, REPUCATION anPRIMING COMPLEX Figure 1.2: A model for replication initiation at 011°C The DnaA protein binds to the four 9-mers, organizing 011°C around the protein core to form the initial complex. DnaA protein then induces duplex opening at the three AT-rich 13-mers forming the open complex. The binding of DnaB- DnaC to 011°C forms the prepriming complex, which is followed by the priming complex and DNA replication (from [30]). 10 open complex. The unwinding of the 13-mers requires the ATP-bound form of DnaA protein and levels of ATP from 1 to 5 mM. Formation of the open complex requires a temperature of at least 38 °C [24] in in vitra assay. The histone-like protein HU or IHF is required for the unwinding of 13-mers by DnaA protein [31]. I After the opening of the 13-mers, DnaB protein complexed with DnaC pro- tein is loaded onto the opened duplex. It has been suggested that DnaA protein guides DnaB protein into the complex [30]. Cross-linking ELISA demonstrated a physical interaction between DnaA and DnaB. This interaction can be inhib- ited with a monoclonal antibody to DnaA protein [27]. The binding of DnaB protein produces a prepriming complex that retains a Pl-sensitive configuration at lower temperature (16°C) [8]. Mapping of the P1 cleavage site showed an ex- tended cleavage pattern compared to that of the open complex. The formation of the prepriming complex requires ATP. dATP and CTP cannot replace ATP [8]. Upon addition of SSB and DNA gyrase and in the presence of ATP, DnaB helicase further unwinds the duplex DNA. The single stranded DNA produced by DnaB helicase is coated and stabilized by SSB protein. The topological strain generated by strand unwinding by DnaB is relieved by DNA gyrase to remove positive supercoils. The extensively unwound structure that is formed, called Form I*, can be separated from other topological forms by agarose gel electrophoresis [29] . Following strand separation and loading of DnaB helicase, primase binds to 11 the single stranded DNA to synthesize RNA primers, thus forming the priming complex. The replication fork contains DnaB helicase, primase and DNA poly- merase III holoenzyme. The moving of the replication fork ultimately results in two daughter chromosomes. 1.2 DnaA protein 1.2.1 dnaA mutants The first temperature sensitive dnaA mutant affecting the initiation of DNA replication CRT46, was isolated and characterized by Hirota et al. [33]. The wild type dnaA gene was then cloned by its ability to complement the tem- perature sensitive phenotype of a dnaA mutant. Its location on the E. coli K-12 linkage map is at 83.5 min, 42 kb counterclockwise from 011°C [34]. The coding region of 1401 nucleotides expresses a basic polypeptide with a deduced molecular weight of 52,633 daltons [36]. Other dnaA alleles have been isolated subsequently [39, 40]. Their respective mutations have been finely mapped by P1 transduction, then at the nucleotide level by DNA sequence analysis [41]. An identical mutation, resulting in substi- tution of alanine to valine at residue 184 very close to the ATP binding site, is found in dnaA5, (11101446, dnaA 601/ 602, and dnaA 604/ 606. These mutants also carry a second mutation of Gly426Ser, Hi5252Tyr, Pr0296Gln, and Ala347Val respectively. The phenotypes of the above mutants are reversible upon temper- ature downshift from nonpermissive to permissive temperature. These mutants 12 reinitiate chromosomal replication in the absence of protein synthesis. All are defective in the proper timing of replication initiation relative to the bacterial cell cycle [41]. Other alleles (dnaA205, dnaA203/204 and (1110.421 1) harbor dif- ferent single mutations that encode substitution in the C-terminal part of the protein corresponding to Val383Met, Ile389Asn, Met411Thr respectively. The mutations of dnaA508 affect Pro28Leu and Thr8011e near the N-terminus. Intragenic suppressors of the temperature sensitive dnaA46 and dnaA 508 alleles have been obtained. These suppressor mutations not only revert the heat sensitivity of the dnaA alleles, but also renders them cold sensitive. The cold sensitive phenotype has been shown to be the consequence of over-initiation that results in lethality. At higher temperature, the mutant protein is partially active, resulting in normal cell growth. 2D protein gel and transcription assay showed that dnaA expression is enhanced in dnaA508003 [37]. The intragenic suppressor of the 1111011508 allele is due to a change of GTG to ATG of the initiation codon that results in elevated expression and apparently accounts for the phenotype of this allele. The dnaA4 6-003 allele encodes two substitutions (Gln156Leu and Tyr271His) in addition to the two mutations present in dnaA46 [38]. Recent biochemical studies demonstrated that over-initiation by the dnaA46cas allele is not caused by protein over-production [53]. Instead, dnaA46cos protein possesses increased replication activity at 30 °C, and retains initiation activity for a longer period in the cell cycle compared to wild type DnaA protein [54]. The systematic study of these suppressor mutations may be of value in understanding the structural 13 and functional relationship of DnaA protein. 1.2.2 Biochemical properties of DnaA protein DNA binding Specific binding of DnaA protein to the E. coli replication origin (011°C) is es- sential for its function as a replication initiator. Supercoiled M13011°C26 DNA containing 0110 was preferentially retained by DnaA protein on a nitrocellulose filter compared to M1301‘iCA221 that lacks the minimal 011°C sequence [58]. If the plasmid was linearized, reduced binding was observed [58], suggesting that DnaA protein binds preferentially to supercoiled DNA. The blockage of the HindIII site located within ariC by preincubating M130riC with DnaA protein further demonstrated that DnaA protein binds specifically to 0110. By compar- ison, the EcaRI site outside the ariC sequence was unprotected [58]. Besides binding to 0110, DnaA protein binds specifically to other DNA fragments in- cluding the replication origins of plasmid pSC101, pBR322, and ColEl; the promoter region of the dnaA gene, the promoter and N-terminal coding region of the miaC gene and a 971 bp Tan fragment of M13 [6]. All these fragments share the common 9 bp DnaA box sequence. DNasel footprinting of DnaA protein at sites containing a single DnaA box showed that the region of protection centers on the 9 bp sequence and extends 40-50 bp to either side [6]. In footprinting of DnaA protein at 011°C contain- ing four DnaA boxes, virtually the entire sequence 5’-TTATCCACA-3’ on one 14 strand is protected, whereas cleavage occurs at the center of the complementary strand [6]. The 9 bp consensus sequence (DnaA box) forms the core for DnaA protein binding. The four DnaA boxes in 0110 designated R1, R2, R3, and R4 presumably act to make 011°C the primary binding target for DnaA protein. In a more recent study on the interaction of DnaA protein with its target recognition sequence, oligonucleotides with sequence variants of the DnaA boxes and with different flanking sequences were examined. DnaA boxes R1 and R4 of 0110 are identical but have different flanking sequences. R2 and R3 differ from R1 and R2 at the fifth and eighth positions, respectively. The equilib- rium dissociation constants and kinetic rate constants were determined. Oligo nucleotides with the sequence TT(A/T)TNCACA are bound by DnaA protein specifically [32]. DnaA boxes R1/R4(TTATCCACA) and R2 (TTATACACA) exhibit specific binding with K D values in the range from 1 to 50 nM. R1 / R4 with its natural 011°C flanking sequence was bound with 50-fold higher afinity than R1/R4 with an averaged flanking sequence. This suggests that flanking sequences affects binding afinity. R3 (TTATCCAAA), R5 (TCATTCACA), and the DnaA box (TTTTCCACA) in the promoter region of miaC gene bind DnaA nonspecifically with K D values greater than 200 nM [32]. The lack of specific binding of R3 in this assay is in agreement with an in viva methylation protection study which showed that DnaA boxes R1, R2, and R4, but not R3 were protected throughout most of the cell cycle [60]. DnaA protein seems to make contact with both the major groove and minor groove [32]. Methyl groups of thymidines are only exposed in the major groove. 15 An oligo nucleotide of DnaA box R4 with all thymidine residues replaced by deoxyuridine in both strands or only one strand was bound with greatly reduced aflinity by DnaA. Pretreatment of the R4 oligo with drugs that bind to the minor groove impaired the binding of DnaA protein in gel retardation assays [32]. Nucleotide binding DnaA protein binds to ATP and ADP with high affinity. The K D values of ATP and ADP binding are 0.03 pM and 0.1 pM respectively [24]. The exchange rate of bound nucleotide with free nucleotide is very slow. Both the stability of DnaA protein and its activities are profoundly affected by nucleotide binding. The binding of ATP / ADP plays an important role in regulating the the initiation of replication through modifying the activity of DnaA protein. Both the ATP-bound form, ADP-bound form, as well asthe nucleotide-free form can bind to 011°C. After binding, strand opening of the 13-mers requires 5 mM level of ATP. Strand separation can be detected by sensitivity to P1 endonuclease. The ADP-form fails to induce a P1 sensitive structure in 011°C. The formation of a prepriming complex, composed of ariC, HU protein, DnaB, and DnaC (as measured by the retention of DnaB in the 011°C complex and replication), is supported by the ATP-form but not the ADP-form of DnaA [24]. Extensive hydrolysis of bound ATP is not essential for the formation of the 16 prepriming complex. Hydrolysis of bound ATP is quite slow (Tl/2:15 min) compared to replication initiation. The non-hydrolyzable analog ATP-75 can replace ATP in forming the prepriming complex [24] . Thus the tightly bound nucleotide seems to serve as an allosteric effector. The tryptic digestion pattern of the ATP- or ADP-form of DnaA protein is distinct compared to that ob- tained with the nucleotide free form [28], suggesting a conformational difference among these forms. The ATP- and ADP-forms of DnaA behave as monomers in solution, while the nucleotide-free form tends to aggregate. This aggregated complex is distinct from the active complexes containing the nucleotide bound form of DnaA [25]. In addition to a domain for high aflinity ATP binding, DnaA protein may have an additional low aflinity ATP binding site. This speculation is based on the requirement of 1-5 mM of ATP for opening the AT-rich region in 0110 by DnaA protein. The subsequent loading of DnaB protein at 011°C to form the prepriming complex requires ATP at 30 [1M [24]. DnaA protein (with its high affinity binding site occupied by ATP) is inactivated by mM levels of ADP in this assay [28]. Presumably, the occupancy of the low affinity binding site by ADP is responsible for the inactivation. The two levels of ATP required for DnaA function may be important for precise regulation of initiation. Interaction °with phospholipids It is well documented that DNA replication in E. 0011' is a. membrane- associated event. Interestingly, disphosphatidylglycerol (cardiolipin) was found to be strik- 17 ingly effective in promoting the rapid release of tightly bound ATP and ADP from DnaA protein [49]. The release of bound ADP by cardiolipin results in the reactivation of the previously inert DnaA protein for replication. The mono- acidic phosphatidylglycerol is only about one tenth as effective as cardiolipin. The neutral phosphotidylethanolamine—the principal E. cali phospholipid, is in- active. Phosphatidylinositol, not present in E. cali, but with the same acidic head group as phosphatidylglycerol, possesses the same activity level. This sug- gests that the acidic head group is important for membrane interaction with DnaA protein. The interaction of DnaA protein with the head group of acidic phospholipids also requires a fluid phase bilayer context. Phospholipids lacking unsaturated fatty acids were relatively inactive compared to those containing unsaturated fatty acids in effecting the release of ADP from DnaA protein [50]. Consistent with these biochemical observations, cells possessing only saturated fatty acids in their membrane due to the inhibition of oleic acid biosynthesis cannot initiate replication, and fail to grow [42]. Cardiolipin inactivates DnaA protein in the absence of bound nucleotide or 011°C [49]. When DnaA protein was purified, half was found in an aggregated form containing phospholipids. The aggregated form was inactive in the recon- stituted replication system. Phospholipase A2 treatment activates aggregated DnaA protein [56]. In vivo evidence also suggests that anionic phospholipids are involved in DnaA-dependent replication from 0110. Mutations in pgsA, which encodes phos- 18 phatidyl-glycerophosphate synthase, lead to arrest of cell growth due to de- fective biosynthesis of phosphatidylglycerol and cardiolipin. Mutations in the mini, which encodes RNaseH, promote chromosomal replication from sites other than ariC thereby bypassing the requirement for the dnaA gene. Mutations in rnhA gene also suppress the growth phenotype of pgsA mutant, suggesting that the block of the pgsA mutation affects initiation at 011C [55]. The interaction of membrane lipids with DnaA protein suggests the involvement of the cell membrane in the regulation of chromosomal replication. 1.2.3 Structural and functional domains of DnaA pro- tein Since the discovery of the E. cali dnaA gene, other bacterial homologs have been identified. In most cases, the gene order dnaA-dnaN—recF—gyrB is conserved. The dnaA gene exhibits a high degree of similarity at the nucleotide sequence level. Amino acid sequence alignment of 14 bacterial homologs reveals conserved residues in a short N-terminal segment and a long C-terminal segment. The amino acid sequence connecting the two conserved segments is not conserved [47]. Thus three structural domains are suggested (Fig. 1.3). Within one of the well conserved regions, a sequence motif GX—X-G-X- G- K-T is present at residues 172 to 179. This consensus sequence is found in many proteins that bind ATP or GTP [44]. Alanine-to-valine at residue 184 is encoded by the (1110.45 and duo/146 alleles. Biochemical characterization of the 19 purified protein demonstrated that both DnaAS and DnaA46 are defective in ATP binding in vitro [45, 52]. Controlled tryptic digestion of DnaA provides some insight into one func- tional domain. Bound to ATP or ADP, a trypsin-resistant 30 kilodalton (kDa) fragment is obtained ([59], Carr and Kaguni, unpublished results). This frag- ment is inactive in 011°C binding but retains the ability of responding to phospho- lipids in releasing tightly bound ATP or ADP [59], suggesting that the functions of high-affinity nucleotide binding and interaction with phospholipids may be in the 30 kDa peptide. In a separate study to identify the DNA binding domain of DnaA protein, different regions of the dnaA gene were amplified and fused to lacZ that encodes fl galactosidase. The DnaA-fl galatosidase fusion was affinity purified by anti- ,6 galactosidase antibody. The fusion containing amino acid residues 374-467 of DnaA protein was sufficient for specific ariC binding in a solid phase DNA binding assay. Fusion to other parts of the dnaA gene, including the N-terminal domain, or the C-terminal domain up to residue 374 did not bind to 011C. Dele- tion of the residues near the boundaries of this 93-amino acid domain (residues 374-467) abolished 011C binding activity. In addition, fusions containing the 93-arnino acid domain from dnaA204 (Ile389Asn), dnaA205 (Val383Met), or dnaA211 (Met411Thr) did not bind to 011°C [51]. Therefore the C-terminal 93-amino acids are considered to be the DNA binding domain. 20 cimASOQ(P28Ll l MSLsLWQQcL AqudELpat ermWIRbLQ aELsdnTLaL yAPNrFVLDW dhaAi08(TBOI) 51 VrdKYLnnIn gLLtchgad apquPevg kpvtqtpqaa vtsnvaapaq 101 vaqtqpqraa pstrsgwdnv papaeptyrs nVNthTFDN FVEGksNgLA dnaA5.dnaA601/602(A184V) dnaA 67(V157G) dnaA46.dnaA604/606(A184V) 1 51 rAAAR N PGgAYNPLFL LLHAVGNgIM arkPNAKVVY 201 MhSERFVqDM VkALQnNaIE EFKrYYRSVD aLLIDDIQFF AnKErSQEBP dnaA46(H252Y) dnaA601/602(P296Ql 251 FNALLEg anIILTSDR YPKEInGVED RLKSRFgWGL tVAIE PELE dnaA6 4/606(A347V) 301 TRVAILmKKA DEndIrLPgE VaFFIAkRLr SNVRELEGAL NR t dnaA205(V383M) 3 51 gramDFVr EaLRnLLalq 7‘"?;.s:“‘?~:”‘w dnaA5 (G4268) dnaA203/204 ( I389N) .e-fi' -- . .77.. .. .\.r,‘, , . . ‘ . ,_ D I o 'rtr'kkif t)“: I I ,._ ,1 pm. 401 ” . ._--_. . .- tf:t£l:l'§'l"[h:l:'\ -_ .-, .7 I -. -....--_.--_.--...-.._. Figure 1.3: Structural and functional domains of DnaA protein The amino acid sequence is of E. coli DnaA protein. Comparison of its amino acid sequence to those of 14 different bacteria revealed the conservation of the amino acid residues [47] (dnaA sequences from other bacteria species are not shown here). Upper case letters indicate that the amino acid is identical or shows a conservative change among 9 bacterial species, bold upper case letters indicate amino acids are identical or show a conservative change among the DnaA protein of 12 out of 14 bacterial species. The region marked “P-loop” denotes the ATP binding site. The boxed region marked “DNA binding” indicates the DNA binding domain. The mutations of the dnaA (TS) alleles are indicated. 21 1.3 Objective of the thesis E. cali DnaA protein consists of a single polypeptide of 467 amino acids with a predicted molecular weight of 52.5 kDa. In its role in initiation of chromo- somal replication, this protein possesses a number of functions, including ATP binding, sequence-specific DNA binding, interaction with DnaB protein and in- teraction with phospholipids. The objective of this thesis project is to correlate its structural domains to specific functions by using a monoclonal antibody ap- proach. This study complements other ongoing projects on DnaA protein in the lab, and provides important reagents that will yield structural information that is currently lacking for DnaA protein. Bibliography [1] Yasuda, S., and Hirota, Y. (1977) Proc. Natl. Acad. Sci. USA 74: 5458-5462. [2] Meijer, M., Beck, E., Hansen, F. G., Bergmans, H. E. N., Messer, W., Van Meyenburg, K., and Schaller, H. (1979) Proc. Natl. Acad. Sci. USA 76: 580-584. [3] Sugimoto, K., Oka, A., Sugisaki, H., Takanami, M., Nishimura, A., Yasuda, Y., and Hirota, Y. (1979) Pro. Natl. Acad. Sci. USA 76: 575- 579. [4] Oka, A., Sugimoto, K., Takanami, M. and Hirota, Y. (1980) Mol. Gen. Gent. 178: 9-20. [5] Asai, T., Takanami, M., and Imai, M. (1990) EMBO J. 9: 4065-4072. [6] Zyskind, R. S., Funnell, B. E., and Kornberg, A. (1984) Cell 38: 889- 900. [7] Matsui, M., Oka, A., Takamami, M., Yasuda, S., and Hirota, Y. (1985) J. Mol. Biol. 184: 529-533. 22 23 [8] Bramhill, D., and Kornberg, A. (1988) Cell 52: 743-755. [9] Hwang, D. S., and Kornberg, A. (1990) Cell 63: 325-331. [10] Kowsalski, D., and Eddy, M. J. (1989) EMBO J. 8: 4335-4343. [11] Sternberg, N. (1985) J. Bact. 164: 490-493. [12] Russell, D. W. and Zinder, N. D. (1987) Cell 50: 1071-1079. [13] Zyskind, J. W., and Smith, D. W. (1992) Cell 69: 5-8. [14] Polacze, P. (1990) New Biol. 2: 265-271. [15] Zyskind, J. W., Harding, H. E., Takeda, Y., Cleary, J. M., and Smith, D. W. (1981) Mol. Cell. Biol. 22: 13-25. [16] Louarn, J ., Funderburgh, M., and Bird, R. E. (1974) J. Bacteriol. 120: 1-5. [17] Bate, D., Asai, T., Cao, Y., Chambers, M., Cadwell, G., Boye, E., and Kogoma, T. (1995) Nucleic Acids Research 23, vol 16: 3119-3125. [18] Landoulsi, A., Malki, A., Kern, R., Kohiyama, M., and Hughes, P. (1990) Cell 63: 1053-1060. [19] Filutowicz, M., Ross, W., Wild, J., and Gourse, R. L. (1992) J. Bact. 174: 398-407. [20] Gille, H., Egan, J. B., Roth, A., and Messer, W. (1991) Nucl. Acids Res. 19: 4167-4172. 24 [21] Oka, A., Sasaki, H., Sugimoto, K., and Takanami, K. (1984) J. Mol. Biol. 176: 433-458. [22] Lu, M., Campbell, J. L., Boye, E., and Kleckner, N. (1994) Cell 77: 413-426. [23] Slater, S., Wold, 8., Lu, M., Boye, E., Skarstad, K., and Kleckner, N. (1995) Cell 82: 927-936. [24] Sekimizu, K., Bramhill, D., and Kornberg, A. (1987) Cell 50: 259-265. [25] Crooke, E., thresher, R., Hwang, D. S., Griffith, J., and Kornberg, A. (1993) J. Mol. Biol. 233: 16-24. [26] Funnell, B. E., Baker, A., and Kornberg, A. (1987) J. Biol. Chem 262: 10327-10334. [27] Marslazek, J ., and Kaguni, J. (1994) J. Biol. Chem. 269: 4883-4890. [28] Yung B. Y., Crook, E., and Kornberg, A. (1990) J. Biol. Chem. 265: 1282-1285. [29] Baker, T. A., Funnell, B. F., and Kornberg, A. (1987) J. Biol. Chem. 262, 6877-6885. [30] Bramhill, D., and Kornberg, A. (1988) Cell 54: 915-918. [31] Hwang, D. S., and Kornberg, A. (1992) J. Biol. Chem. 267: 23083- 23086. 25 [32] Schaper, S., and Messer, W. (1995) J. Biol. Chem 270: 17622-17626. [33] Hirota, Y., Mordoh, J ., and Jacob, F. (1970) J. Mol. Biol. 53: 369-387. [34] Hansen, F. G., Koefoed, S., Sorensen, L., and Atlung, T. (1987) EMBO J. 6: 255-287. [35] Zyskind, J. W., Cheary, J. M., Brusiolw, W. S. A., Harding, N. E., and Smith, S. W. (1983) Proc. Natl. Acad. Sci. USA 80: 1164-1168. [36] Hansen, F. G., Hansen, E. B., and Atlung, T. (1982) EMBO J. 1: 1043-1048. [37] Eberle, H., Van de Merwe, w., Madden, G. K., Wright, L. and Donlon, K. (1989) Gene 84: 237-245. [38] Braun, R., O’Day, K. and Wright, A. (1987) J. Bact. 169: 3898-3903. [39] Abe, M., and Tomizawa, J. (1971) Genetics 69: 1-15. [40] Wehr, C. T., Waskell, L., and Glaser, D. A. (1975) J. Bact. 121: 99107. [41] Hansen, F. G., Koefoed, S., and Atlung, T. (1992) Mol. Gen. Gent. 234: 14-21. [42] Fralick, J. A., and Lark, K. G. (1973) J. Mol. Biol. 80: 459-475. [43] Holz, A., Schaefer, C., Gille, H., Juterbock, W. R., and Messer, W. (1992) Mol. Gen. Gent. 233: 81-88. 26 [44] Saraste, M., Sibbald, P. R., and Wittinghofer, A. (1990) Trends Biochem. Sci. 15: 430-434. [45] Hupp, T. R., and Kaguni, J. (1993) J. Biol. Chem. 268: 13128-13136. [46] Ogden, G. B., Pratt, M. J., and Schaechter, M. (1988) Cell 54: 127-135. [47] Skarstad, K. and Boye, E. (1994) Biochemica et Biophysica Acta 1217: ‘ 111-130. [48] Kaguni, M. J ., and Kornberg, A. (1984) Cell 38: 183-190. [49] Sekimizu, K., and Kornberg, A. (1988) J. Biol. Chem. 263: 7131-7135. [50] Yung, B. Y., and Kornberg, A. (1988) Proc. Natl. Acad. Sci. USA 85: 7207-7205. [51] Roth, A., and Messer, W. (1995) The EMBO Journal 14: 2106-2111. [52] Hwang, D, S., and Kaguni, M. J. (1988) J. Biol. Chem 263: 10633- 10540. [53] Katayama, T., and Kornberg, A. (1994) J. Biol. Chem 269: 12698- 12703. [54] Katayama, T. (1994) J. Biol. Chem. 269: 22075-22079. [55] Xia, W., and Dowhan, W. (1995) Proc. Natl. Acad. Sci. USA 92: 783- 787. 27 [56] Hwang, D. S., Crooke, E., and Kornberg, A. (1990) J. Biol. Chem. 265: 19244-19248. [57] Hwang, D. S., and Kornberg, A. (1992) J. Biol. Chem. 267: 23087- 23091. [58] Fuller, R. S., and Kornberg, A. (1983) Proc. Natl. Acad. Sci. USA 80: 5817-5821. [59] Yung, B. Y., and Kornberg, A. (1988) J. Biol. Chem. 264: 6146-6150. [60] Samitt, C. E., Hansen, F. G., Miller, J. F. and Schaechter, M. (1989) EMBO J. 8: 989-993. Chapter 2 Epitope mapping and functional analysis of monoclonal antibodies to DnaA protein 2.1 Introduction DnaA protein of E. cali consists of a single polypeptide chain of 467 amino acids with a predicted molecular weight of 52.6 kDa [5]. DnaA protein is required for the initiation of chromosomal replication from 011°C [23, 24, 4]. At the step of initiation of replication, DnaA protein recognizes and binds to the four 9-bp consensus sequences (DnaA boxes) in 0110. In the presence of 1-5mM ATP, binding induces a localized duplex opening at the AT-rich 13-mer region [46]. Subsequently, DnaA protein is proposed to guide DnaB helicase (complexed with DnaC) to bind to 011°C through a physical interaction between DnaA pro- tein and DnaB protein [22]. DnaB helicase further unwinds the duplex and guides primase to synthesize primers on the DNA template [27]. The elonga- tion of primers by DNA polymerase III holoenzyme eventually results in two 28 29 daughter chromosomes. Biochemically, DnaA protein possesses several activities important for its physiological role as a replication initiator and regulator. First, DnaA protein binds DNA specifically. It recognizes and binds to DNA containing the sequence TTAT(A/C)CA(A/C)A [7]. Second, it binds to ATP and ADP with high affin- ity. The ATP-bound form is active and the ADP-bound form is only partially active in forming replication complexes [29]. Third, it interacts with phospho- lipids. This interaction promotes the release of the tightly bound nucleotide [6]. The fast release of ADP rejuvenates inert DnaA protein by allowing it to bind ATP. Fourth, it interacts directly with DnaB protein [22]. Its structural complexity is suggested by the multiple functions of the protein. Sequence com- parison of dnaA homologs among fourteen different bacterial species [1] reveals that DnaA protein at the amino acid level is highly conserved within a short N-terminal domain and a longer C-terminal domain. The phenotype of tem- perature sensitive dnaA mutants exhibits some correlation with the locations of mutations, suggesting the existence of specific domains that relate to specific functions. At present, little information is known concerning fImctional domains of DnaA protein. It has been proposed that the consensus sequence motif GX4GKT (P-loop) at residues 172-179 (reviewed in [2]) is involved in high aflinity ATP binding [29]. Proteins encoded by the (Ina/146 and dnaA5 alleles, both of which contain identical amino acid substitutions of alanine 184 to valine (A184V), as well as other unique substitutions, are unable to bind ATP in vitra [2, 3]. 30 In a recent study, the mutant protein containing only the A184V substitution was studied biochemically and shown to be defective in ATP binding (K. M. Carr and J. M. Kaguni in press). The DNA binding activity has recently been localized to a 93-amino acid C-terminal domain [32]. Other domains of DnaA protein involved in low affinity ATP binding, interaction with phospholipids and interaction with DnaB protein are unidentified. Monoclonal antibodies can be a useful tool to correlate the structure of a protein to its various functions. Utilizing this approach, monoclonal antibodies against DnaA protein were generated and characterized for their inhibitory ef- fects on several activities of DnaA protein, including replication initiation, DNA binding, ATP binding, F1* formation and the interaction with DnaB protein. In the studies reported here, epitopes for these monoclonal antibodies were pre- cisely mapped by constructing and screening a peptide library derived from the dnaA gene. Reported below, some antibodies inhibit replication of plas- mids containing 011°C but not of an M13 derivative containing a DnaA protein binding site in a proposed hairpin structure [44]. Priming of DNA replication in vitra on the latter template, termed as ABC priming, involves assembly of an intermediate formed on the single-strand (ss) DNA that is dependent on DnaA protein. These results suggest that a subset of DnaA protein functions are required for ABC priming compared to those involved in replication of 011°C plasmids. The region of DnaA protein bound by M7 antibody that interferes with the interaction between DnaA and DnaB protein [22] has been identified. Presumably, a domain in this region interacts directly with DnaB- protein. Re- 31 sults with a third class of antibodies suggest that DnaA protein may act at a later step in the initiation process, perhaps through interaction with subunits of DNA polumerase III holoenzyme. 2.2 Experimental Procedures Reagents, Proteins, and DNAs: Commercial enzymes and proteins were from the following sources: bovine serum albumin, DNase I, Sigma; T4 DNA polymerase, Tth DNA polymerase, T4 DNA ligase, linearized pTOPE—lbp, and competent HMSl74 (recAI hst 111‘) lysogenized by ADE3, Novagen; Sequenase, USB. Highly purified replication proteins were: DnaA protein (fraction IV, 2 x 105 units/mg) [4]; DnaB protein (fraction V, 6 x 105 units/mg) [8]; DnaC protein (fraction VI, 3 x 106 units/mg) [9]; primase (fraction V, 2 x 106 units/mg) [10]; single-stranded DNA binding protein (SSB) (fraction IV, 4 x 104 units/mg) [11, 12]; DNA polymerase III holoenzyme (fraction V, 2 x 105 units/mg) [13]; DNA gyrase A subunit (fraction III, 2 x 105 units/mg), and DNA gyrase B subunit (fraction V, 1 x 105 units/mg) [14]; RNA polymerase (fraction V, 250 milliunits/mg) [15, 16]; topoisomerase I (fraction III, 5 x 104 units/mg) [17]; RNase H (fraction IV, 8x 105 units / mg) [18], and HU protein (fraction IV, 5x 10“ units/ mg) [19]. Units for the above proteins are described in the corresponding references. Monoclonal antibodies to DnaA protein were produced [17] and purified from 32 tissue culture supernatants with protein A or Mono-Q Superose (Pharmacia). Unless otherwise indicated, the monoclonal antibodies were in 40 mM Hepes- KOH pH 8.0, 50 mM KCl, 15% glycerol, and 2 mM dithiothreitol (DTT) by dialysis. Monoclonal antibody 2B to rat brain hexokinase, and goat anti-mouse antibodies that are specific for each immunoglobulin subclass were gifts from Professor John Wilson, Michigan State University. Antisera specific for DnaB protein was obtained from rabbits [17]. M13-A site ssDN A [12] contains a DnaA protein recognition sequence (DnaA box) in the stem of a hairpin structure formed by base pairing of an inverted repeat from the R6K 'y-origin region but is not contained in the minimal DNA fragment identified as the 7-origin. M1301‘iC2LB5 contains E. cali 0110 [16]. Epitope Mapping: Libraries of recombinant plasmids, each of which ex- pressed a small peptide derived from DnaA protein as a fusion protein joined to T7 gene 10 protein, were constructed essentially as described by the Novagen. The dnaA gene was amplified by PCR, treated with DNaseI in the presence of Mn“, and fragments averaging 50-150 base pairs in size were purified from an agarose gel. The DNA fragments were then treated successively with T4 DNA polymerase and Tth DNA polymerase to end-fill and then add a single dA residue to the 3' end of each fragment, followed by ligation to linearized pTOPE-lb with a single dT over hang at each 3’ end, and transformation into HMSl74(z\DE3), both obtained from the manufacturer. Colonies transferred to nitrocellulose filters were lysed with chloroform vapor, and the filters were placed on Whatman 3MM paper saturated with 20 mM 'I‘ris-HCl pH7.9, 6 M 33 urea, and 0.5 M NaCl. Positive clones were identified by immunoblot analy- sis with the monoclonal antibodies of interest. Detection of antibody-antigen complexes was with horseradish peroxidase conjugated to goat anti-mouse IgG (Bio-Rad). After color development, the filters were aligned with the original plate, the positive clones were colony purified and verified as immunoreactive by the above method. Plasmid DNA, isolated by an alkaline lysis procedure, was sequenced by the enzymatic method with primers that flank the site of insertion of the vector. The epitope was deduced by comparative analysis of the DNA sequences of inserts. The cross-reactivity of A22 and M7 with dnaA nonsense mutants The dnaA+ gene and an ocher mutant at codon 184 in pACYC184 were from this laboratory (M. D. Sutton and J. M. Kaguni, manuscript in preparation). Whole cell lysates were prepared by resuspending of 108 cells of the plasmid bearing strain (Sup‘ ) collected at mid-log phase in 0.1% sodium dodecyl sulfate (SDS) and electrophoresed on 10% SDS-polyacrylamide gels. The protein was then transferred to PVDF membranes (Schleicher & Schuell). A22, M7 and M48 were used as primary antibodies and goat anti-mouse IgG HRP conjugate was used as the secondary antibody. ECL chemiluminescence kit (Amersham) was used to detect antibody-antigen complexes. DNA Binding Assays: Fragment retention assays (25 [11) contain 6 ng of a 459 bp Sall-Xhal fragment containing 0110 from pT80182 DNA and 100 ng of Hinfl-digested pBR322 DNA as a nonradioactive competitor in buffer containing 40 mM HEPES-KOH (pH 7.8), 5 mM MgC12, 2 mM DTT, and 50 34 mM KCl. 3° end-labeling of the 011°C fragment was performed with the large fragment of DNA polymerase I, and [a—32P] dATP. Monoclonal antibodies (160- 250ng) were added to DnaA protein (80ng) and incubated on ice for 15 min to assay for inhibition on DNA binding activity. DNA was then added and reactions were incubated at 30°C for 10 min, the reactions were filtered through nitrocellulose filters (Millipore HAWP, 0.22 pm, 24 mm) and washed with 250 pl of the above buffer equilibrated at room temperature. Radioactivity retained on the filters was determined by liquid scintillation counting. Gel mobility shift assays [20] were performed by addition of the indicated amounts of DnaA protein and 1.48 pg of HaeIII-digested M1301'iC2LB5 DNA in 25 pl containing 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgC12, 10% (v/v) glycerol, and 2 mM DTT. Monoclonal antibodies were added to DnaA protein and incubated for 15 min on ice to assay their inhibitory effects. After incubation at 30 °C for 10 min, the samples were electrophoresed on 1% agarose gel in 90 mM Tris-borate, and 1mM EDTA. DNA bands are visualized by staining with ethidium bromide. ATP Binding Assays: Reactions (25pl) were performed essentially as described [29] and contained equal amounts (2 pmol) of DnaA protein and monoclonal antibodies, and 5 mM DTT, 15% glycerol, 0.01% Triton X100, and 50 mM 'I‘ris-HCl pH 8.0. Incubation was for 15 min on ice. 0.1 pM [ac—32F ATP] (0.5 pCi) were then added and incubation was continued at 0 °C for 15 min followed by filtration through nitrocellulose filters (Millipore HAWP, 0.22 pm, 13 mm). The filters were then washed with 500 pl of the above buffer at 35 room temperature. Radioactive ATP retained on the filters was quantified by liquid scintillation counting. Replication Assays: Reaction mixtures for ABC priming (25 pl) were assembled essentially as described in [18] and contained 40 mM HEPES-KOH pH 8.0 (measured at 1 M and 20 °C), 40 mM potassium glutamate, 10 mM magnesium acetate, 4 mM DTT, 0.1 mg/ml bovine serum albumin (BSA), 4% (w/v) sucrose, 2 mM ATP, 0.25 mM each of CTP, GTP, and UTP, 100 pM each of dATP, dCTP, dGTP, and [methyl-3H]dTTP (25-30 cpm/pmol), 0.1 pg M13-A site ssDNA, 1 pg SSB, 28 ng DnaA protein, 50 ng DnaB protein, 24 ng DnaC protein, 10 ng primase, and 80 ng DNA polymerase III holoenzyme. Reaction mixtures were assembled at 0 °C, then incubated for 15 min at 30 °C for DNA synthesis. To measure the inhibitory effect, DnaA protein was pre-incubated with monoclonal antibodies, then added to the reaction. Total nucleotide incorporation (in pmol) was measured by liquid scintillation counting after trichloroacetic acid precipitation (TCA) onto glass fiber filters (Whatman GF/ C). Reactions of 011°C plasmid replication (25 pl) were similar to those described in [2] and were comparable to those for ABC priming except for the following: CTP, GTP, and UTP were at 0.5 mM each; phosphocreatine, 6mM; creatine ki- nase, 100pg/ml; M1301'iCZLBS supercoiled DNA instead of M13-A site ssDNA, 200 ng; SSB, 160 ng; HU, 25 ng; gyrase A subunit, 470 ng; gyrase B subunit, 600 ng; DnaA protein, 56 ng; DnaB protein, 55 ng; DnaC protein, 24 ng; primase, 10 ng; and DNA polymerase III holoenzyme, 80 ng. Reactions were assembled 36 at 0 °C and incubated for 30 min at 30 °C to measure DNA synthesis. Assay of FI“ formation: Single stage reactions (25 pl) contain 40 mM HEPES-KOH (pH 7.6), 20 mM Tris-HCl pH 7.5, 4% (w/v) sucrose, 2 mM ATP, 4 mM DTT, 11 mM magnesium acetate, 200 ng M13 011°C2LB5 supercoiled DNA, 21 ng HU protein, 160 ng SSB, 100 ng DnaB protein, 24 ng DnaC protein, 500 ng gyrase A subunit, 1 pg gyrase B subunit, and 60 ng DnaA protein. Monoclonal antibodies were added to DnaA protein and followed by incubating on ice for 15 min to assay their inhibitory effects. Incubation was at 30 °C for 25 min. Reactions were stopped by addition of SDS to 2.5%, and EDTA to 10 mM. The samples were electrophoresed at 25 V for 18 h in 1% agarose gels in 90 mM Tris-borate pH 8.3, and 1 mM EDTA. DNA was detected by ethidium bromide staining. Two stage reactions (25 pl) were performed essentially as described in [21] and contained 30 mM HEPES-KOH pH 8.0, 0.4 mg/ml BSA, 20% glycerol, 5 mM EDTA, 6 mM 08.012, 0.4 mM ATP, 80 ng M1301‘iC2LB5 DNA, and the above proteins. Monoclonal antibodies were added to DnaA protein and incubated for 15 min on ice. Reactions were incubated for 30 min at 30 °C, then placed on ice to add 500 ng gyrase A subunit, 1 pg gyrase B subunit, magnesium acetate to 10 mM, and ATP to 2mM. Incubation followed for 10 min at 24 °C. NagEDTA to 50 mM and SDS to 2.5% were added and electrophoresed as above. Enzyme-linked Immunosorbent Assay (ELISA): DnaA protein (0.5pg / well) or BSA (10pg / well) was added to 96 well microtiter trays (Nunc-Immuno 37 Plate, Maxisorp, Inter-Med) in 50 pl of buffer containing 0.137 M N aCl, 2.7 mM KCl, 10 mM N agHPO4, and 1.76 mM KH2P04 pH 7.3 (PBS). Incubation was for 1h at room temperature. Unbound proteins were removed with four successive washes (200 pl each) of PBS containing 0.2% (w/v) BSA. The last wash was incubated for 1 h before removal. The indicated monoclonal antibodies diluted in PBS containing 0.2% BSA was added, incubated for 1 hour, and followed by three washes to remove unbound antibodies. DnaB protein (30 ng in 50 pl of PBS containing 0.2% BSA) was then added and incubated for 15 min. Glutaraldehyde (2 pl of a 2.5% solution in water) was added to 0.1%, followed by incubation for 30 min. The wells were then washed with 200 pl of PBS containing 25 mM Tris-HCl pH 7.5, and 0.2% BSA once, then three times with 200 pl of PBS containing 0.2% BSA. Rabbit antisera to DnaB protein at 10“ fold dilution in 100 pl PBS containing 0.2% BSA was added, incubated at °C overnight, and followed by three washes to remove unbound antibodies. Antibody-antigen complexes were detected after incubation for 1 h with goat anti-rabbit IgG horseradish peroxidase conjugate. Colorimetric detection was with O-phenylenediamine (0.4 mg/ ml) and hydrogen peroxide (0.3% v/v) in 100 pl of 50 mM sodium citrate, pH 4.0. After addition of 4 N sulfuric acid, absorbance Was measured at 490 nm with a BioTek EL 310 plate reader. 38 2.3 Results 2.3.1 Identification of continuous epitopes To identify the epitopes recognized by monoclonal antibodies to DnaA protein, a peptide library was constructed by fusing DNA fragments produced by DNase I treatment of the dnaA gene to part of T7 gene 10 encoding the N-terminal 260 amino acids. Expression of the fusion protein depends on the T7 RNA polymerase gene under lac U V5 promoter control. Transformants (2-5x 103 per antibody) were replicated onto nitrocellulose filters, lysed with chloroform va- por, then screened with each antibody to identify immunoreactive clones. In addition, transformants recognized by one antibody were tested for reactivity with other appropriate antibodies. For example, transformants bound by A3 were also found to react with M100 (Table 1). Similarly, M12 and M36 bound to the same set of transformants. Clone 7 was obtained by its immunoreactiv- ity to a monoclonal antibody not described here and was characterized early in this work. Transformants of clone 7 were bound by antibodies M85, M48, M43, M100 and M1. Table 1 listed the immunoreactivity of antibodies to various recombinants that were useful in defining the minimal epitopes. This approach is designed to identify continuous epitopes. It also assumes that amino acids from T7 gene 10 protein do not contribute to the epitopes. Immunoreactive clones were obtained with most antibodies except for A22, and M7. The portion of DnaA protein in the fusion was deduced by DNA sequence analysis of inserts in recombinant plasmids. M1, M10, M12, M36, 39 M43, M48, M85 recognize linear epitopes located between residues 125-146. A3 and M100 recognize residues 104-114 and 110-114, respectively. The epitope for M60 resides in residues 86-98. 40 Table 2.1: Deduced epitopes of monoclonal antibodies to DnaA protein Monoclonal Antibodies Immunoreactive Clonesa Amino Acid Residues Deduced l'ipitopesb M36 M36-3 Ill-148 M48-4 111-146 M48-5 125-148 125-146 M12 M48-4 Ill-146 M48-5 125-148 125- 146 M36-3 Ill-148 M85 M85-l 120- 154 M85-3 101-144 M85-12 118-138 133-138 Clone-7 133- 147 M48 M48-4 111-146 M48-5 125-148 Clone-7 133-147 133-146 M43 M43-1 131-146 M43-3 131-146 M43-4 111-141 133-141 Clone-7 133-147 M10 M10-6 113-146 M10-7 120-142 133-142 Clone-7 133-147 M1 M48-4 111-146 M48-5 125-148 133-146 Clone-7 133-147 A3 A3-2 94-117 A3-1 84-114 104-114 M100-4 104-119 M100 M100-4 104-119 M100-6 110-130 M100-9 101-126 110-114 M100-11 110-127 A3-l 84-114 A3-2 94-117 M60 M60-2 86-98 M60—5 70-100 8698 M60-6 76-112 M7 M36-3 111-148 111-148 41 Continuation of Table 2.1 aThe nomenclature of an immunoreactive transformant correlates the antibody that originally identified it. bDeduced epitopes are indicated relative to the amino acid sequence of DnaA protein. 6Amino acids from residues 125-146 are EPTYRSNVNVKHTFDNFVEGKS, from 104-114 are TQPQRAAPSTR, from 86—98 are TPQAAVTSNVAAP. 42 2.3.2 A22 and M7 appear to recognize conformational epitopes Immunoblot experiments indicated that A22, M7, and M48 were comparably immunoreactive to full length DnaA protein. However, A22 was not reactive to transformants of Table 1 that expressed fusion proteins of T7 gene 10 and portions of dnaA gene (data not shown). M7 was only reactive to the trans- formant M36-3. No positive clones for A22 were identified after screening over 2 x 104 transformants. By comparison, about 2 positive clones were obtained for each 103 transformants screened with antibodies that appear to recognize linear epitopes, generally considered to be composed of 4-8 amino acids [45]. Based on this reasoning, about 40 positive clones were expected if A22 recognizes a continuous epitope. As recombinant plasmids were constructed to contain 50- 150 bp of the dnaA gene, these results suggest that the epitope recognized by A22 resides in a longer region and may be conformational. A collection of nonsense mutants of the dnaA gene have been obtained (M. Sutton and J. Kaguni, manuscript in preparation). In a non-suppressing strain, one encodes a truncated polypeptide of 147 amino acids, established as an ochre mutant by DNA sequence analysis and confirmed by size by SD S-polyacrylamide gel electrophoresis relative to molecular weight standards. To localize the por- tion of DnaA protein bound by A22 and M7, immunoblot analysis was per- formed with a nonsuppressing strain bearing this plasmid-encoded mutant as well as other mutants with nonsense codons located more distantly. As a con- trol, M48, whose epitope resides within residues 133-146, recognizes the 147 43 residue long ochre peptide. M7 and A22 bind to the 147 amino acid long ocher peptide (Fig. 2.1) and longer nonsense peptides (data not shown). However, the response of M7 to the 147 amino acid peptide was much weaker than to the full length DnaA protein (Fig. 2.1). This result suggests that the epitope recognized by M7 is not entirely in the first 147 amino acids, or that its conformation after transfer results in less binding. Whereas these results suggest that the epitopes recognized by A22 and M7 are conformational and in the N-terminal region, they are not identical. First, the response of A22 to the ochre peptide was stronger than that of M7 (Fig. 2.1). Second, by immMoblotting M7 bound to the fusion protein encoded by plasmid M36-3 (encoding residues 111-148 of DnaA protein) whereas A22 did not. M7 and A22 also bound to a polypeptide (data not shown) which is encoded by an in-frame deletion mutant that lacks of amino acids 220-294 (J. Lipar and J. M. Kaguni, unpublished results). By comparison, neither antibody was immunoreactive with transformants containing plasmid M85- 12 (encoding a fusion protein containing residues 118-138 of DnaA protein), M48-5 (residues 125-148), clone 7 (residues 133-147), M10-5 (residues 237-245), M10-6 (residues 113-146), M10-7 (residues 120-142), M62 (residues 86-98), or M60-6 (residues 76-112). The lack of immunoreactivity with these recombinants suggests that the epitope recognized by M7 and contained in plasmid M36-3 is within residues 111-148. The epitope recognized by A22 appears to be within the first 147 residues. 44 M48 A22 M7 is: is is ‘5' Bl bl 2% 2% 2% 522 is: 52: 02200.9. 00.0. J" 16kDa — . . - Figure 2.1: Monoclonal antibody M7 and A22 recognize conformational epitopes Whole cell lysates were prepared from E. cali HMSl74 harboring the dnaA+ gene (pACYCdnaA) or an ochre mutant at codon 148 (pACHA-22) in pACYClS4 (M. Sutton, and J. M. Kaguni, manuscript in preparation). Purified DnaA protein (5 ng) served as a control for immunoblot analysis with the indicated monoclonal antibodies. In lanes containing the ochre peptide, the immunoreactive species at the position of full-length DnaA protein is chromosomally encoded. We do not know the identity of other reactive species. In this experiment, the difference in mobility of the ochre peptide in the immunoblots of Fig. 2.1 may be due to dissimilar electrophoretic conditions, despite precautions to treat each gel identically. (This experiment was performed by C. Margulies who was a coworker on this project) 45 2.3.3 DNA binding is not inhibited by monoclonal an- tibodies Filter binding assays with a labeled restriction fragment containing the 011°C sequence reveal that none of the antibodies substantially reduced DNA binding (data not shown, summarized in Table 2.3). At a saturating level of DnaA protein and in the absence of antibody, about 5.2 ng of the 6 ng 011C fragment was retained. Reactions containing antibodies resulted in retention of 4.8-5.9 ng of DNA. The failure to detect inhibition of DNA binding was regardless of the order of addition of DnaA protein, and antibodies. Fragment mobility shift assays were also performed with a restriction diges- tion of a ariC-containing plasmid. Addition of M100, M85, and A3 as well as the remaining antibodies did not affect the DNA binding activity of DnaA protein measured by this assay (data not shown). Under these experimental conditions, the 011°C fragment did not migrate as a discrete complex when bound by DnaA protein. Binding was inferred by the reduction in the level of unbound ariC fragment. 100 g «8351- N 25 :< 2:222 Antibody 2.0 — ATP (mM) 0.5 Figure 2.2: DNA binding activity is not inhibited by monoclonal antibodies Reactions (see “Experimental Procedures”) contained the indicated amount of antibodies and DnaA protein. Antibodies were incubated with DnaA protein to measure their effects on DNA binding. (This experiment was performed by J. Marszalek who was a coworker on this project.) 47 2.3.4 ATP binding is not inhibited by monoclonal an- tibodies DnaA protein binds to ATP with high aflinity (KD of 0.03 pM). The ATP- bound form is active in replication. Comparative sequence analysis revealed a conserved P-loop motif (GX4GKT) found in many nucleotide binding proteins. The effect of monoclonal antibodies on ATP binding was examined to determine if any were inhibitory. M85 did not inhibit ATP binding (Fig. 2.1). None of other antibodies were found to reduce ATP binding activity by greater than 15% under conditions in which 0.3 ATP were bound per monomer of DnaA protein (summarized in Table 2.3) 2.3.5 Several monoclonal antibodies inhibit 011°C repli- cation and ABC priming Six monoclonal antibodies inhibited DNA replication of an 0110 containing plas- mid (Fig. 2.4). M7 and A22 inhibited to near background levels at a ratio of two DnaA protein molecules per antibody molecule. By comparison, inhibition by M1, A3, M100, and M60 required higher antibody levels. The remaining antibodies were not inhibitory (data not shown). As the inhibitory antibodies did not appear to interfere with binding to DNA or ATP, inhibition was for another cause. 48 ,4 05 < a - I. C o 0.4 '- a/N\a .- 3 . g. . = 0.3 " - O E 3 a 0.2 - - l- . 4 g 0.1 - - 3 O m 0.0 I A l a l a T 0 250 500 750 M85 (119) Figure 2.3: M85 do not inhibit the ATP binding activity of DnaA protein DnaA protein (2 pmol) was incubated with increasing amount of M85 at 0 °C for 15 minutes, then 0.2 pM (a—32P) ATP was added. After 10 min incubation on ice, the reaction products were filtered through nitrocellulose filters and radioactivity retained on the filters were counted. 49 The monoclonal antibodies were also tested for inhibition of ABC priming. This assay measures the replication activity of DnaA protein with a single- stranded M13 derivative harboring a DnaA box in a proposed hairpin struc- ture. DNA synthesis also requires single strand DNA binding protein, DnaB, and DnaC, primase and DNA polymerase III holoenzyme. In contrast to the an- tibodies that inhibit 011°C replication, only M7 and A22 inhibited ABC priming substantially (Table 2.2). Other antibodies were only slightly inhibitory (data not shown). Previous studies showed that M7 interfered with the interaction between DnaA protein and DnaB protein in DnaB-DnaC complex only if M7 was added to DnaA first [22]. A22 may inhibit ABC priming by a similar mech- anism as inhibition was dependent on the order of addition (Table 2.2). Other antibodies that inhibit 011°C replication do not inhibit ABC priming appreciably whether added before or after the BC complex (data not shown). 50 Table 2.2: The influence of monoclonal antibodies on ABC priminga-b Incubation B Incubation A DNA Synthesis(pmol) Relative Activity None DnaB+DnaC 243 100% M7 DnaB+DnaC 21 8 DnaB+DnaC M7 18 1 74 None DnaB+DnaC 23 1 100 A22 DnaB+DnaC 90 40 DnaB+DnaC A22 175 77 Incubation A Incubation B DNA synthesis(pmol) Relative Activity None DnaB+DnaC 230 100% Ml DnaB+DnaC 207 90 DnaB+DnaC M1 185 80 A3 DnaB+DnaC 193 84 DnaB+DnaC A3 221 96 M60 DnaB+DnaC 260 1 13 DnaB+DnaC M60 240 104 a Reactions assembled on ice contained M13 A-site 55 DNA, ribo- and deoxy-ribo- nucleotides, magnesium acetate and DnaA protein at amounts used for ABC priming (see "Experimental Procedures"). The indicated monoclonal antibodies (100 ng each), DnaB, and DnaC protein (as indicated, 50 and 24 ng each, respectively) were added in incubations (A or B), each for 10 min at 30 0C. DNA synthesis was then measured after addition of primase (10 ng) and DNA polymerase III holoenzyme (80 ng) and incubation at 30 0C for 30 min. bThese experiments were performed by Jarek Marszalek who was a coworker on this project. 51 0 4080120160 ,_.. M100 3 3m)- e- g 200- I!) o 5 100- I: a o . . g o 100 200 300 a antibody (ng) Figure 2.4: Inhibition of 011°C replication by monoclonal antibodies. Reactions were assembled as described in “Experimental Procedures” and con- tained the indicated amounts of monoclonal antibodies. (This experiment was performed by J. Marszalek who was a coworker on this project.) 52 2.3.6 Antibody inhibition of DNA unwinding by DnaA protein It is possible that the antibodies which specifically inhibit 011°C replication block one or more functions of DnaA protein not essential for ABC priming. Bound to 0110, DnaA protein induces a localized unwinding at the three AT rich 13-mers [46], followed by binding of DnaB helicase to unwind further the parental duplex. Unwinding is detected by its sensitivity to a ssDNA specific nuclease [46, 26]. Alternatively, addition of DNA gyrase to remove the positive superhelicity in the DNA generated by DnaB helicase results in a highly negatively supercoiled DNA [21]. This topological form, termed F1*, migrates more rapidly on an agarose gel than the supercoiled plasmid DNA isolated from E. cali. The inhibition of 0110 replication activity by M1, A3, M100, and M60 may be due to inhibition of unwinding activity that is not expected to be required for ABC priming. To test this possibility, these and other antibodies were examined for their effects on the unwinding activity of DnaA protein. In an assay involving addition of DNA gyrase in a second stage of incubation, M7 and A22 were marginally inhibitory (data not shown). The remaining antibodies were not. This assay was simplified to involve one incubation instead of two. ATP was sufficient at 0.5 mM or greater with a minimal incubation of 10 min (data not shown). Under these conditions, M7 was inhibitory at 0.5 or 2 mM ATP, whereas inhibition of by other antibodies (A22, M100, and A3) was more effective at the higher concentration (Fig. 2.5). This finding suggests that incubation of DnaA protein with 2 mM ATP may induce a conformation more favorable for antibody 53 M100 M85 A3 4— o -><- 50-> §§ +220->0 <125> §§ Antibodymg) o 45 90180 0 45 901809090 0 45 90180 0 45 901909090 DnaA(ng) 4 oriC Figure 2.5: M7, A3, M100 and A22 inhibit F1* formation at 2 mM ATP. Reactions contained M7, 250 ng; A3, 250 ng; M100, 200ng; or A22, 160 ng; and ATP as indicated in a one stage incubation. (This experiment was performed by J. Marszalek who was a coworker on this project.) 54 binding. It also supports the suggestion that a low affinity ATP binding site exists [46] in addition to the site that confers high affinity ATP binding. The greater inhibition observed at 2 mM ATP may also explain the modest inhibition observed in the two stage incubation in which ATP was at 0.4 mM during this stage. Inhibition by A22 and M100, as well as M7 and A3 was proportional to the amount added (data not shown). The remaining antibodies were not inhibitory even at 5-fold higher levels than those used here. That M1 and M60 inhibited ariC replication, but ABC priming only poorly, and failed to inhibit F1* formation, suggests that they affect an unknown activity of DnaA, perhaps subsequent to unwinding. 2.3.7 Antibodies inhibit the interaction between DnaA and DnaB DnaA protein has been shown to interact physically with DnaB protein by use of an ELISA assay [22]. In this method, immobilized DnaA protein is incubated with either DnaB alone, or as a complex with DnaC protein. The complex of DnaB bound to DnaA protein is stabilized by glutaraldehyde cross-linking. Crosslinking of DnaC protein to DnaA and BSA was not observed . M7 inhibited the interaction between DnaA and DnaB in this assay (Fig. 2.8), this result is consistent with previous results [22]. Examination of other monoclonal antibodies indicated that they are also inhibitory (Figs. 2.7, 2.8). As a control, a monoclonal antibody to rat brain hexokinse had little effect on the binding of DnaB protein to immobilized DnaA protein (Fig. 2.6). Whereas 55 the ELISA method demonstrated a specific physical interaction between the two proteins, it failed to correlate the inhibition of replication with the inhibition of DnaB binding. Inhibition of the interaction between DnaA and DnaB may be due to steric hindrance. For example, binding of a divalent antibody to im- mobilized DnaA protein may produce a network that occludes binding of DnaB protein. Fab fragments may correlate inhibition of replication to inhibition of DnaB binding, but this has not yet been tested. 56 .° 01 P or .° a. ° —9— DnaA-M85 " —+— DnaA-283 —I— DnaA-No antbody P 00 .° N ADSOI’DHI‘ICO at 490 nm 9 I 4 I n I e l e I em 0 200 400 .600 800 10001200 MAb(ng) .0 c Figure 2.6: Monoclonal antibody 2B (against hexokinase) does not inhibit the interaction between DnaA and DnaB. DnaA protein (0.5 pg/ well) or BSA (10 pg/ well) was added to microtiter plates. Monoclonal antibody 23 or M85 was then added at the indicated amounts, followed by incubation for 1 h to allow binding. Unbound antibodies were removed by three successive washes with PBS containing 0.2% BSA. 50 ng of DnaB protein was added and allowed to bind for 15 min. After cross-linking with glutaraldehyde, the unbound protein was removed and DnaB protein was detected by ELISA with rabbit anti-DnaB antisera. 57 1'2 _ . -—-G— No antibody-DnaA _ _ —o— M1-DnaA E 1'0 _ —-— M10-DnaA O 08 - -' _'°—' M12-DnaA a, . V . —l‘— M36-DnaA as 05 - " —D— M48-DnaA 3 . - ——*— M100-DnaA g 0.4 ~ - —a— mom 3 * ' —I- A4-DnaA 3 0.2 - ‘ —+—' A22-DnaA < ' ° —-I-— No antibody-BSA 0.0 DnaB (ng) Figure 2.7: Monoclonal antibodies M1, M10, M12, M36, M48, M100 and A3 inhibit the interaction between DnaA and DnaB protein. DnaA protein (0.5 pg/ well) or BSA ( 10 pg/ well) was added to microtiter plates. Monoclonal antibodies at the indicated amounts were then added, followed by incubation for 1 h to allow binding. Unbound antibodies were washed away by three successive washes with PBS containing 0.2% BSA. The indicated amounts of DnaB protein were added and allowed to bind for 15 min. After cross-linking with glutaraldehyde, the unbound protein was removed and DnaB protein was detected by ELISA with rabbit anti-DnaB antisera. 58 ‘ —°— Mt-BSA ' ‘_"— Ml-DnaA ‘ ‘—"—' MZDnaA - —'°— M10-DnaA - """—' M43-DnaA - —O— M85-DnaA —*— M100-DnaA Absorbance at 490 nm MAb(ug) Figure 2.8: Monoclonal antibodies M1, M7, M10, M43 and M85 interfere with the interaction between DnaA and DnaB proteins. DnaA protein (0.5 pg/ well) or BSA ( 10 pg/well) was added to microtiter plates. Monoclonal antibodies were then added at the indicated amounts, followed by incubation for 1 h to allow binding. Unbound antibodies were removed by three successive washes with PBS containing 0.2% BSA. 50 ng of DnaB protein was added and allowed to bind for 15 min. After cross-linking with glutaraldehyde, the unbound protein was removed and DnaB protein was detected by ELISA with rabbit anti-DnaB antisera. 59 Table 2.3: Summary of the linear epitopes and the inhibitory effects of mono- clonal antibodies to DnaA protein Monoclonal Epitope ariC ABC priming F1* DNA ATP antibodiesb (a.a.) replication replication formation binding binding M36, 1ng}, 125-146 - - - - - M12, IgG. 125-146 - - - - - M85, IngB 133-138 - - - - - M48, IgG1 133-146 - - - - - M43, IgG] 133-141 - - - - - M10, IgG3 133—142 — - - - - A3, IgG] 104-114 + - + - _ M100,Ig61 110-114 1» - + - _ M l , IgG3 133-146 + - - - _ M60, IgG3 86-98 + - - - _ M7, IgGZA confro. +++ +4—1- +++ - - A22, IgG1 confro. ++ -H- ++ - - aoriC replication and F l * formation was performed by a Jarek Marszalek who was a coworker on this project bThe monoclonal antibodies were classified by an ELISA method with preparations of goat anti-mouse antibodies that are specific for each immunoglobulin subclass. C-I-I-+ very strong inhibitory ++ strong inhibitory - not inhibitory 60 2.4 Discussion In order to correlate the structure of DnaA protein to its various functions, the inhibitory effect of monoclonal antibodies to DnaA protein was character- ized. The epitopes for these monoclonal antibodies were precisely mapped by constructing and screening a peptide library derived from portions of the dnaA gene. The epitopes were deduced from the DNA sequence of the inserted frag- ment of recombinant plasmids isolated from positive clones. Most antibodies appear to recognize linear epitopes located within a small region near the N- terminus. The clustering of epitopes suggests that this region is highly antigenic, and surface exposed in the native conformation of DnaA protein. The epitopes of M7 and A22 are conformational and reside in longer amino acid sequences. None of the antibodies inhibit DNA binding, which suggests that the region containing various epitopes (residues 86- 146) is not involved in DNA binding. This finding is consistent with the observation that the C-terminal 93 amino acid residues are responsible for DNA binding activity. First, fusion protein containing the C-terminal region of DnaA protein from residue 379 to the end are suflicient for 011C DNA fragment binding [32]. Second, nonsense and missense mutants that affect the C-terminal region of DnaA protein are defective in DNA binding (M. D. Sutton and J. M. Kaguni, manuscript in preparation) In DnaA protein, the P-loop motif, GX4GKT located at residues 172-179, is found in many ATP binding proteins (reviewed in [28]). The structure of the P-loop has been determined by X-ray crystallographic analysis of adenylate 61 kinase [34, 35], elongation factor 'I‘u [36], RecA [30] and ms 21 protein [31]. In ms 21, the conserved residues interact with the ’y-phosphate of the bound nucleotide and with a magnesium ion that chelates the B— and 7—phosphates [31]. Mutagenic analysis of RecA protein [37, 38] supports the conclusion that the corresponding residues interact with the a- and fl-phosphates of ATP. None of the antibodies described here inhibit high affinity ATP binding. This result is consistent with the location of respective epitopes that do not overlap the P-loop motif. In addition, the residues bound by respective antibodies are not apparently involved in interacting with adenine of ATP nor does antibody binding appear to interfere sterically or to alter the conformation of the ATP binding domain of DnaA protein. Inhibition by M7 and A22 Antibodies M7 and A22 inhibit DnaA protein in 011°C replication, ABC priming, and F1* formation involving an unwinding of the AT-rich region by DnaA protein. A critical event in each of these reactions involves a direct interaction between DnaA and DnaB protein. M7 had pre- viously been shown to inhibit the binding of DnaB protein (from DnaB-DnaC complex) to a replication intermediate of DnaA protein bound to 011C [22]. By a modified ELISA procedure, M7 was shown to interfere with the binding of DnaB protein to immobilized DnaA protein [22]. The epitope recognized by M7 is within residues 111-148, whereas the epitope bound by A22 is within the first 147 amino acids. These findings suggest that residues within the M7 epitope may be involved in the interaction between DnaA protein and DnaB protein. Antibody binding can also induce a conformational change [39, 40]. An 62 alternative possibility that we can not exclude is that the binding of M7 induces a conformational change that interferes with the interaction between these two proteins. Inhibition by A3 and M100 High affinity binding of ATP to DnaA pro- tein is essential for its activity in inducing duplex opening at the AT-rich 13- mers in 011°C [29, 41]. By comparison, the ADP-bound form of DnaA protein is relatively inert. Because unwinding is not required in ABC priming, both nucleotide bound- forms are active in ABC priming [44]. A3 and M100 inhibit 011°C replication and F1* formation, but ABC priming only poorly. These ob- servations suggest that A3 and M100 inhibit 011°C replication by affecting the unwinding activity of DnaA protein. If so, residues bound by A3 and M100 are involved in unwinding. Inhibition by M1 and M60 The initiation process at 011C involves three consecutive steps. First, DnaA protein in the ATP-bound form binds to the DnaA boxes. Second, DnaA protein induces the localized duplex opening at the AT-rich 13-mers. Third, DnaB protein binds to the 0110 through a direct interaction with DnaA protein. The least understood result in this study is the inhibition pattern of M1 and M60. The two antibodies inhibit 011°C replication, yet do not inhibit ABC priming and F1* formation. The ABC priming assay reflects DnaA protein’s activity of binding to DNA and DnaB protein. F1* formation additionally measures the unwinding activity of DnaA protein. The lack of effect of these antibodies in these two assays suggests inhibition of an activity subsequent to unwinding. No known biochemical activity of DnaA 63 protein has been identified at a step after unwinding and interaction with DnaB protein. However, genetic studies of extragenic suppressors of dnaX that map to the dnaA gene [42, 43] suggest an interaction between DnaA protein and the dnaX gene products, 1' and 7, which are components of DNA polymerase III holoenzyme. However, the extragenic suppressors of dnaX have been character- ized as missense and nonsense mutations at residues 213, 432, and 435 of DnaA protein [43]. This result is inconsistent with the epitopes bound by M1 and M60. Another inconsistency is that other antibodies bind to the same epitope as M1 are not inhibitory. This may be explained by a specific conformational change induced by M1 binding to result in inhibition. It is also possible that the actual epitope for M1 is a few residues different from that of the other monoclonal antibodies although the epitopes for all of them have been mapped in the same " region. Further investigation is required to substantiate whether DnaA protein possesses other novel activities that are revealed by M1 and M60 antibodies. Bibliography [1] Skarstad, K., and Boye, E. (1994) Biochemica et Biophysica Acta 1217: 111-130. [2] Hwang, D. S., and Kaguni, J. (1988) J. Biol. Chem. 263: 10633-10640. [3] Hupp, T. R., and Kaguni, J. (1993) J. Biol. Chem. 268: 13128-13136. [4] Fuller, R. S., and Kornberg, A. (1983) Proc. Natl. Acad. Sci. USA 80: 5817-5821. [5] Hansen, E. B., Hansen, F. G., and Von Meyenburg, K. (1982) Nucleic Acids Res. 10: 7373-7385. [6] Sekimizu, K., and Kornberg, A. (1988) J. Biol. Chem. 263: 7131-7135. [7] Fuller, R. S., Funnell, B., and Kornberg, A. (1984) Cell 38: 889-900. [8] Arai, K., Yasuda, S., and Kornberg, A. (1983) J. Biol. Chem. 256: 5247-5252. [9] Kobori, J ., and Kornberg, A. (1982) J. Biol. Chem. 257: 13763-13769. 64 65 [10] Wold, M. S., and Mcmachen, R. (1982) Proc. Natl. Acad. Sci. USA 79: 4907-4911. [11] Weiner, J. H., Bertsch, L. L., and Kornberg, A. (1975) J. Biol. Chem. 250: 1972-1980. [12] Chase, J. W., Whittier, R. F ., Auerbach, J., Sancar, A., and Rupp, W. D. (1980) Nucleic Acids Res. 8: 3215-3227. [13] McHenry, C., and Kornberg, A. (1977) J. Biol. Chem. 252: 6478-6484. [14] Mizuuchi, K., Mizuuchi, M., O’Dea, M. H., and Gillert, M. (1984) J. Biol. Chem. 259: 9199-9210. [15] Burgess, R. R., and Jendrisak, J. J. (1975) Biochemistry 14: 4634-4638. [16] Gonzalez, N., Wiggs, J ., and Chamberlin, M. J ., Arch. Biochem. Bio- phys. (1977) 182: 404-408. [17] Kaguni, J. M., and Kornberg, A. (1984) J. Biol. Chem. 259: 8578-8583. [18] Naito, S., Kitani, T., Ogawa, T., Okazaki, T., and Uchida, H. (1984) Proc. Natl. Acad. Sci. USA 81: 550-554. [19] Dixon, N. E., and Kornberg, A. (1984) Proc. Natl. Acad. Sci. USA 81: 424-428. [20] Parada, C., and Marians, K. J. (1991) J. Biol. Chem. 266: 18895-18906. [21] Baker, T. A., Sekimizu, K., Funnell, B. E., and Kornberg, A. (1986) Cell 45: 53-64. 66 [22] Marszalek, J., and Kaguni, J. M. (1994) J. Biol. Chem. 269: 4883-4890. [23] Kohiyama, M. (1968) Cold Spring Harb. Symp. Quant. Biol. 317-324. [24] Hirota, Y., Ryter, A., and Jacob F. (1968) Cold Spring Harb. Symp. Quant. Biol. 677-693. [25] Bramhill, D., and Kornberg, A. (1988) Cell 49: 743-755. [26] Hwang, D. S. and Kornberg, A. (1992) J. Biol. Chem. 267: 23038- 23086. [27] Tougu, K., Peng, H. and Marians, K. J (1994) J. Biol. Chem. 269: 4675-4682. [28] Messer, W. and Weigel, C. (1996) in Escherichia cali and Salmonella typhimurium. Cellular and Molecular Biology (Neidhardt, F. C., In- graham, J. L., Low, K. B., Magasanid, B., Dchaechter, M., et al eds) vol. 2, American Society for microbiology, Washington, D. C. [29] Sekimizu, K., Bramhill, D. and Kornberg, A. (1987) Cell 50: 250-265. [30] Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsh, W., et al (1990) EMBO J. 9: 2351-2359. [31] Story, R. M. and Steitz, T. A. (1992) Nature 355: 374-376. [32] Roth, A. and Messer, W. (1995) EMBO J. 14: 2106-2111. [33] Masai, H., Nomura, N. and Arai, K. (1990) J. Biol. Chem 265: 15134- 15144. 67 [34] Muller, C. w. and Schulz, G. E. (1992) J. Mol. Biol. 224: 159-177. [35] Dreusicke, D., Karplus, P. A. and Schulz, G. E. (1988) J. Mol. Biol. 199: 359-371. [36] Jurnak, F. (1985) Science 230: 32-36. [37] Logan, K. M. and Knight, K. L. (1993) J. Mol. Biol. 232: 1048-1059. [38] Konola, J. T., Logan, K. M. and Knight, K. L. (1994) J. Mol. Biol. 237: 20-34. [39] Colman, P. M., Laver, W. G., Varghese, j. N ., Baker, A. T., Tulloch, P. A., Air, G. M. and Wevster, R. G. (1987) Nature 326: 358-363. [40] Stanfield, R. L., Fieser, T. M., Lerner, R. A., and Wilson, I. A. (1990) Science 248: 712-719. [41] Yung, B. Y. and Kornberg, A. (1989) J. Biol. Chem. 264: 6146-6150. [42] Walker, J. R., Ramsey, J. A. and Haldenwang, W. G. (1982) Proc. Natl. Acad. Sci. USA 79: 3340-3344. [43] Gines, C. E., Blinkova, A. and Walker, J. R. (1995) J. Bacteriol. 177: 705-717. [44] Masai, H., Nomura, N. and Arai, K. (1990) J. Biol. Chem. 265: 15134- 15144. [45] Pinilla, C., Appel, J. R. and Houghten, R. A. (1993) Mol. Immunol. 30: 577-585. 68 [46] Bramhill, D. and Kornberg, A. (1988) Cell 52: 743-755. Chapter 3 Summary and Perspective The replication of the E. cali chromosome begins at a unique site, 011C, and is a precisely regulated event in the bacterial cell cycle. Regulation occurs at the time of initiation of chromosomal replication. Of over 20 genes whose products participate in this process, the dnaA gene that encodes DnaA protein is unique because of its central role in initiation. Because of this and because DnaA protein may regulate the process, it has been the focus of study in a number of laboratories. Biochemical approaches have revealed that DnaA protein is multi-functional. First, it is a sequence-specific DNA binding protein, recognizing and binding to 9-mer sequences, termed as the DnaA boxes. DnaA protein binds to the four DnaA boxes in 0110 in an ordered manner. Upon binding, it induces a localized duplex opening at the AT-rich 13-mers near the left boundary of 011C. It is proposed to guide the binding of DnaB helicase to 0110 through a direct physical interaction with DnaB protein. DnaA protein also binds ATP and ADP with high affinity. The ATP-bound form is active in inducing duplex 69 70 unwinding whereas the ADP-bound form is relatively inert. Acidic membrane phospholipids interact with DnaA protein to promote the rapid release of tightly bound ATP or ADP. DnaA protein is ubiquitous among bacterial species. Sequence comparison of dnaA homologs revealed that conserved sequences are mainly in a short N- terminal domain and a long C-terminal domain. The multiple functions of DnaA protein suggest that it may be structurally composed of several domains, each participating in a specific biochemical function required for its activity in DNA replication. In order to correlate the structure of DnaA protein to specific functions, mon- oclonal antibodies to DnaA protein were produced, and the inhibitory effects on activities of DNA binding, ATP binding, unwinding of 011C, and replication were characterized. None of the antibodies inhibited DNA binding and ATP binding. Monoclonal antibodies M7 and A22 inhibited replication of an 0110 plasmid, ABC priming, and the formation of a highly unwound 011°C plasmid (Form 1*) that is dependent on the initial unwinding of the 13-mers at the left boundary of 011C by DnaA protein. Monoclonal antibodies A3 and M100 in- hibited oriC replication and F1"' formation. M1 and M60 inhibited only 0110 replication. The epitopes for these monoclonal antibodies have been determined by constructing and screening a DnaA peptide library. Monoclonal antibodies M1, M10, M12, M36, M43, M48, M60, M85, M100, and A3 recognize contin- uous epitopes clustered at the same region (from residues 86 to 148) of DnaA protein. M7 and A22 recognize conformational epitopes within the N-terminal 71 147 amino acids. These studies suggest that amino acids 86-148 are not involved in DNA binding and ATP binding, in agreement with other studies that localize the DNA binding domain to the C-terminal 93 amino acid residues, and the ATP binding domain to residues 172-179, containing a P-loop motif found in many nucleotide binding proteins. Theses studies also suggest that regions of DnaA protein are involved in interaction with DnaB protein, and in either unwinding of 011°C, or low-affinity binding of ATP. DnaA protein may also interact with subunits of DNA polymerase III holoenzyme. Most of the monoclonal antibodies bind to linear epitopes located in a region encompassing amino acid residues 86 to 148. This feature that limits the corre- lation of structure to functional activities of DnaA protein can be overcome by generating other monoclonal antibodies that bind to different regions of DnaA protein. The region of DnaA protein involved in the interaction of acidic phos- pholipids has not been determined. Characterization of these antibodies as well as others (not yet prepared) that bind elsewhere may be useful in identifying the domain involved in this function. Findings from this study may relate to the role of the cell membrane in DNA replication. That E. cali DNA replication is membrane-associated has been well documented but the molecular basis is not understood. An ELISA assay was used to measure the interaction between DnaA and DnaB protein. With this assay, all antibodies inhibited the binding of DnaB to immobilized DnaA protein, whereas only a few inhibited the replication activity 72 of DnaA protein. Of the latter class, M7 was shown to inhibit binding of the DnaB-DnaC complex to DnaA protein bound to an 011°C plasmid, or M13 containing a DnaA box motif in a hairpin structure. Apparently, the monoclonal antibodies that did not inhibit replication may block the binding of DnaA to DnaB for steric reasons by ELISA. Because the molecular weight of an IgG molecule is about 3 times larger than that of DnaA protein, the use of smaller Fab fragments in the assay might allow us to correlate directly the inhibition of replication activity with the inhibition of DnaB binding. The inhibition patterns of M1 and M60 suggest that DnaA protein is proba- bly involved in replication at a step after unwinding and interacting with DnaB protein. Genetic studies suggest that DnaA protein interacts with the subunits of DNA polymerase III holoenzyme. Immunoprecipitation and cross-linking ELISA can be used to investigate further the interaction of DnaA protein with other replication proteins including subunits of polymerase III holoenzyme, SSB, Primase, DNA gyrase, RNA polymerase and others. "‘Illllllllllll“