W 3 ‘ ! 'L! Ksfi (O J —| CD "6/: 2 .h ‘4 00 “D OVERDUE FINES: .‘.________.__ ‘ 25¢ per day per Item RETURNING LIBRARY MATERIALS: ______._._._._____________ p lace in book return to remove charge from circulation records CELL DIVISION IN CIS:PLATINUM(II)DIAMMINODICHLORIDE- INDUCED FILAMENTS OF ESCHERICHIA COLI: DEPENDENCE UPON COMPLETION OF DEOXYRIBONUCLEIC ACID REPAIR AND A PROTEASE ACTIVITY By Bruce Edward Markham A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1980 ABSTRACT CELL DIVISION IN ClSyPLATINUM(II)DIAMMINODICHLORIDE- INDUCED FILAMENTS 0F ESCHERICHIA COLI: DEPENDENCE UPON COMPLETION OF DEOXYRIBONUCLEIC ACID REPAIR AND A PROTEASE ACTIVITY By Bruce Edward Markham The inorganic antitumor compound gjsfplatinum(lI)diammino- dichloride (PDD) induced extensive filamentation in wild-type Escherichia coli and in mutants lacking certain deoxyribonucleic acid (DNA) repair functions including 333A, [£28, rggC, and 991A. Wild type, 991A and rggBC filaments were observed to fragment with increasing periods of incubation after removal of PDD while 335A filaments failed to divide when observed microscopically on agar slides. Washed filaments of these strains were all capable of fragmenting in broth cultures when they were held in phosphate buffer at 4C for at least 4 h prior to incubation. A majority of the filaments regained colony forming ability during incubation in liquid medium subsequent to the removal of P00. Alkaline sucrose gradient analysis of DNA from filaments revealed the presence of single strand gaps resulting both from excision repair and replica- tion over DNA-POD adducts. Completion of gap repair, as monitored by increasing sedimentation rate in alkaline sucrose gradients of Bruce Edward Markham template and newly synthesized DNA after PDD removal, coincided with the time of the first microscopically observed fragmentation event. DNA synthesis resumed near the time of initiation of frag- mentation. At concentrations which had no effect on cell growth, or DNA degradation, the protease inhibitor antipain prevented PDD- induced filamentous growth of wild type strain N3llO. This compound delayed fragmentation of wild type filaments when added to cultures subsequent to removal of PDD. Antipain had no effect on DNA repair. The results are consistent with the hypothesis that completion of DNA repair, return of the duplex to a supercoiled conformation, and subsequent protolytic breakdown of a division inhibitor are required for the reinitiation of cell division. ACKNOWLEDGMENTS I would like to express my sincere gratitude to Dr. Robert R. Brubaker, my major advisor, for his guidance, consideration, and sense of humor throughout my graduate study. Thanks is also extended to Dr. Doris Beck for her generous donations of the platinum compound, helpful discussions, and moral support; to Dr. L. Snyder and Dr. P. T. Magee for helpful suggestions and to the members of my guidance committee. A special thank you is extended to my wife, Gwen Markham, who put up with the trials and tribulations of being a graduate student's wife and for her support. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ORGANIZATION Section I. LITERATURE REVIEW . Biological Effects of PDD Cell Division . . . . . . Constituitive DNA Repair Processes The SOS Hypothesis . . Literature Cited II. FRAGMENTATION OF CIS:PLATINUM(II)DIAMMINODICHLORIDE- INDUCED FILAMENTS OF WILD TYPE AND DNA REPAIR- DEFICIENT MUTANTS OF ESCHERICHIA COLI Introduction . . . Methods and Materials Results Discussion III. INFLUENCE OF CHROMOSOME INTEGRITY 0N CELL DIVISION IN CIS;PLATINUM(II)DIAMMINODICHLORIDE-INDUCED FILAMENTS OF WILD TYPE AND DEOXYRIBONUCLEIC ACID- REPAIR DEFICIENT MUTANTS OF ESCHERICHIA COLI . Materials and Methods Results Discussion . Acknowledgments Literature Cited Page vi viii 57 62 88 91 92 Section IV. EFFECT OF THE PROTEASE INHIBITOR ANTIPAIN 0N RECOVERY OF DIVISION POTENTIAL IN CIS- PLATINUM(II)- DIAMMINODICHLORIDE- INDUCED FILAMENTS OF ESCHERICHIA COLI K-lZ Abstract . Literature Cited Acknowledgments iv Page 96 98 113 115 Table LIST OF TABLES Section II PDD induces filamentation in DNA repair-deficient mutants of E, coli . . . . . . Fragmentation of PDD induced filaments Section III Bacterial strains Page 55 56 66 Figure LIST OF FIGURES Section I A model for the role of [ggA protein X in the induc- tion of SOS functions Section II The effect of cis- -platinum(II)diamminodichloride on the viability of cultures . . . Rescue of colony-forming ability by filaments . Net synthesis of deoxyribonucleic acid in filaments . Sedimentation profiles in alkaline sucrose gradients of deoxyribonucleic acid released from filaments of E, coli W3llO . Sedimentation profiles in alkaline sucrose gradients of deoxyribonucleic acid released from filaments of E, coli ABZSOO Sedimentation profiles in alkaline sucrose gradients of deoxyribonucleic acid released from E. coli W3llO and ABZSOO after growth for one doubling period withIQig- platinum(II)diamminodichloride . Sedimentation profiles in alkaline sucrose gradients of deoxyribonucleic acid isolated from E. coli W3llO grown with and without cis- -platinum(II§- diamminodichloride . . . . . . . . Sedimentation profiles in alkaline sucrose gradients of deoxyribonucleic acid released from wild type, png, recB, C, and uvrA isolates of E. coli K-lZ after growth with cis- -platinum(II)diammino- dichloride . . . . . . . vi Page 30 68 71 74 76 78 81 83 86 Figure Section IV Increase in optical density at 620 nm of E. co oli W3llO incubated with and without antipaTn Colony-forming ability of E. coli W3110 incubated with and without antipain . . . . . . Profiles of alkaline sucrose gradients of deoxy- ribonucleic acid from E, coli W3llO grown with PDD and/or antipain . . . . . . . Colony-forming ability of PDD-induced filaments in the presence or absence of antipain Profiles on alkaline sucrose gradients of deoxy- ribonucleic acid from filaments of E, coli W3llO grown with PDD, then incubated in fresh medium with antipain . vii Page 100 102 105 108 111 ORGANIZATION This thesis is organized in such a way as to fulfill the departmental requirement for a publishable manuscript as well as to provide the reader with background information to aid in understand- ing the implications of results presented. Section I consists of a review of the literature pertinent to the research discussed. Section II is a published manuscript describing initial observations on fragmentation of PDD induced filaments. This work was presented at a satellite symposium of the Seventh International Congress of Pharmacology: Coordination Chemistry and Cancer Chemotherapy, Toulouse, France meeting, July 1978. Sections 111 and IV are manu- scripts prepared for submission to the Journal of Bacteriology. viii SECTION I LITERATURE REVIEW Biological Effects of PDD Bacteria PDD was first isolated from growth medium during an investi- gation on the effects of an electric field on growth processes in E, gglj_(l72), andidentified as one of the agents responsible for the production of long multinucleated filaments (169, 171). Fila- mentous growth was produced when other gram negative bacteria were treated with PDD (135). Division of PDD-induced filaments was initiated by removal of the compound, but not by treatment with divalent cations, pantoyl lactone, or elevated temperatures. Gram positive bacteria were resistant to the activity of this compound, and filamented slightly at near toxic levels of the drug (169). 191Pt-labeled PDD was associated In radioactive tracer studies, with protein, nucleic acids and metabolic intermediates in lysates of E, gglj_filaments, but only with proteins from cells which had been killed prior to PDD treatment (l56). A similar compound gj§f dichloro-([G-3H]-dipyridine)-platinum(II) bound strongly to DNA and RNA from various sources but not with the proteins examined. Sodium chloride inhibited this binding which suggested that dis- sociation of chlorine atoms from PDD was necessary for its binding activity (92). In a comparative study on the effects of PDD and mitomycin C on UV sensitive E, 9911, a strong correlation was observed between the decrease in colony forming ability and inhibition of DNA syn- thesis with increasing concentrations of PDD. Protein and RNA synthesis were only secondarily affected. Mitomycin C was more potent than PDD in its effect on survival and DNA synthesis. The mode of action of PDD, mitomycin C and UV light were suggested to be similar (l8l). This conclusion was supported by the observation that growth of E. ggli lambda lysogens in the presence of PDD resulted in induction of the prophage (l57). Indirect induction of prophage occurred when PDD treated F+ donor strains were mated with F' recipient lysogens (158). The DNA repair capacity of E, £911 appears to be crucial to the maintenance of colony forming ability when cells are exposed to PDD. Shimizu and Rosenberg (lBl) observed that DNA repair- deficient 85-1 and 85-2 mutants were more sensitive to PDD than their parental strains. Sensitivity to PDD was enhanced 2 to 5 or 13 to 23 times in flg§_and g§:_mutants respectively (44). The rate of killing caused by PDD was substantially greater in mutants defi- cient in excision and recombination repair than in their repair- proficient counterparts (4). PDD induced a high frequency of mutagenesis in Salmonella typhimurium TA98 or TAlOO, and in E, coli. Base substitution and frame shift mutations induced by PDD in E, typhimurium depended on the presence of an R-factor plasmid carrying information for error-prone DNA repair (1). E, coli cells, mutated with agents whose mutagenic mechanism of action was known, were treated with PDD, and the frequenty of reversion of selected mutations indicated that PDD primarily promoted base substitutions. PDD reversion of frame shift mutations was observed at a reduced frequency (5). Eukaryotic Cells PDD selectively inhibited DNA synthesis in cultured human amnion AV 3 cells, Erlich ascites tumor cells, and Chinese hamster cells (72, 91, 209) with RNA and finally protein synthesis affected at higher concentrations. DNA and RNA synthesis in lymphocytes stimulated with phytohemagglutinin were also inhibited when treated with POD (93). Pascoe and Roberts (147) compared the amount of PDD bound to macromolecules with its cytotoxic effect on Hela cells. 1 survival, when the approximate At a dose of P00 resulting in e- molecular weight of the macromolecules was taken into account, many more platinum molecules were bound per DNA molecule than to RNA or protein. The effectiveness of P00 in inducing interstrand cross-links was shown to be approximately 5 times greater jg_xi!g than 1Q vitro. Vanden Berg and Roberts (208,209) reported that Chinese hamster cells were capable of repairing interstrand cross-links induced by PDD within 21 hours after treatment. Analysis of DNA on alkaline sucrose gradients, subsequent to P00 treatment, revealed that all DNA sedimented in the region of 7005 at 2h, 3505 at 6h and at the near normal 400-6505 at 21h. The narrow range of DNA sedimentation values at 6h, coupled with results from previous DNA-PDD binding studies, suggested that only inter- strand cross-links and not single strand damage was repaired by the cells excision repair processes. The PDD induced inhibition of DNA synthesis in Chinese hamster cells was reversed by the addi- tion of caffeine (210); however, DNA synthesized under these con- ditions was found to be of a lower molecular weight than that from untreated control cells. The reduction in molecular weight was directly proportional to the dose of PDD administered. Newly synthesized DNA was of a size approximately equal to the inter- platinum distance on the template strand suggesting the presence of gaps opposite PDD-DNA products. When caffeine was removed from the culture, there was a rapid increase in the molecular weight of nascent strands. Inhibition of postreplication repair was proposed to account for this effect. PDD induced mutations in the hypoxanthine-guanine phos- phoribosyl transferase (HGPRT) locus of Chinese hamster cells have been reported in several studies (144, 207). Mutations in this gene resulted in cell resistance to the cytotoxic purine analogues 8-azaguanine and 6-thioguanine. The high frequency of UV-induced mutations in the HGPRT locus of DNA repair-deficient human skin fibroblast cells derived from patients with xeroderma pigmentosum led to the conclusion that mutations arose from unrepaired DNA 1esions (121). PDD induced mutations in this same cell system (V. Maher, personal communication). Leopold et al. (112) observed the induction of sarcomas in rats and mice by PDD. These results suggested that treatment of patients with antitumor platinum complexes may impose the risk of induction of second tumors in long term survivors. Deoxyribonucleic Acid Selective inhibition of DNA synthesis by PDD treatment (72, 91, 181) and the correlation between cytotoxicity and DNA binding (147, 148) indicated that DNA was the target of PDD activity. Changes in the ultraviolet absorption spectrum of various nucleo- sides and calf thymus DNA provided evidence that PDD binds to purine and pyrimidine bases (81, 123). Treatment of calf thymus DNA with PDD jg_yj§§9_facilitated renaturation which suggested that inter- strand DNA cross-links had formed holding both strands in register during denaturation (71, 81). Subsequently DNA cross-links were identified in POD treated Hela cells. Comparative studies on DNA binding of P00 and its less cytotoxic, non-antitumor, Egggg isomer failed to show a quantitative relationship between cytotoxicity and crosslinking (147). These results were supported by similar results with gig: and Eggg§¢p1atinumlv compounds (148). Shooter et al. (193) reported that interstrand cross-links were not the cause of inactivation of bacteriophage T7 and R17. They compared phage inactivation with PDD, and various mono and bifunctional alkylating agents. Inactivation occurred exclusively with PDD or bifunctional aklylating agents, but at concentrations insufficient to produce significant interstrand cross-linking. PDD inactivation of transforming DNA was observed (140). This evidence suggested that the inactivation of these biological processes by PDD was not the direct result of interstrand cross-links. Intrastrand cross- linking of neighboring bases was proposed to be the biologically significant event. Robbins (160,161) demonstrated that radioactive platinum(II) compounds preferentially bound to guanine moieties in DNA. PDD blocked the G+GATCC specific sequence cleavage of DNA by the restriction endonuclease Bam-l suggesting that intrastrand cross- links between adjacent guanine residues had occurred (101). PDD binding to DNA resulted in an increase in the partial specific volume and in the electrophoretic mobility of DNA molecules examined (29, 194). These results were interpreted to indicate interruption of base pairings by PDD, resulting in local regions of single strand DNA, which under conditions of low ionic strength, collapse and reduce the effective size of the molecule. Antitumor Activity PDD had potent antitumor activity against Sarcoma 180 and Leukemia L 1210 while the Ergg§_iosmer of this compound was rela- tively ineffective (170, 173). Inorganic platinum compounds repre— sented a new class of antitumor agents. The broad range of tumors against which PDD was found to have some activity has recently been reviewed (163). Human tumors proven to be most sensitive to P00 include testicular carcinoma, ovarian adenocarcinoma, squamous cell carcinomas, endometrial carcinomas, malignant lymphomas, and head and neck cancers. Enhanced antitumor activity of P00 has been observed when used in combination with other drugs and radiation. PDD was recently approved by the F.D.A. for clinical treatment of certain tumors and is marketed under the name of cisplatin (B. Rosenberg, personal communication). Clinically significant side effects from PDD treatment included myelosuppression, nephro- toxicity, audiological impairment and severe nausea and vomiting (206). Although PDD was found to be an immunosuppressant (102, 229), a role for the immune response in PDD mediated activity was suggested. Mice cured of advanced Sarcoma 180 rejected all attempts to reintroduce tumors. Preincubation of tumor transplants with PDD inhibited the ability of the tumor to grow in the mice, but these animals were immune to further transplantation. Introduction of dead tumors failed to induce this immunity, indicating that PDD enhanced antigenicity of tumor cells (166). Combined treatment of 5-180 tumors with PDD and the immunostimulant zymosan, resulted in a two fold increase in the cure frequency (32). Although supportive evidence for this model is insufficient, mitotically inhibited enlarged tumor cells which result from PDD treatment may exhibit enhanced antigenicity (163). The antitumor activity of P00 was proposed to depend on the gig configuration of the molecule since the Eggfl§_isomer was found to be relatively inactive against tumors. Since only £j§_compounds have the potential to chelate, the antitumor activity was proposed to be largely associated with a PDD-DNA chelation reaction (28). P00 formed bidentate chelates at various positions on adenosine, guanine and cytidine (123, 167), however, no correlation between specific binding sites and antitumor activity has been determined. The most recent model proposed by Rosenberg (168) for PDD induced antitumor activity is based on the assumption that sensitive tumor cells are deficient in the repair of PDD-DNA lesions. In DNA repair-deficient tumor cells, miscoding platinum DNA lesions pro- mote a mutation opposite these lesions which results in the eventual death of the tumor cell. Cell Division Cell division in Escherichia coli appears to result from a complex series of coordinated events, requiring the integrated regulation of many cellular activities. Evidence suggests that division is related to DNA replication (26, 40, 42, 73-73, 77, 124), DNA repair (18, 19, 53, 90, 223), synthesis of proteins during and after completion of rounds of DNA replication (23, 95, 100, 140) and changes in the cell envelope (95, 96, 99, 180). Chromosome Replication and Cell Division The observation that cell division was blocked when DNA synthesis was inhibited, but that a block in cell division did not necessarily inhibit DNA synthesis suggested a regulatory connection between these two activities (124). A temporal relationship between these functions was established by Helmstetter et a1. (76). The time for a round of DNA replication (C), the time between termina- tion of replication and subsequent cell division (0) and the sum (C+D) was quantified over a wide range of growth rates. At growth 10 rates of one doubling time per hour or less, the values of these parameters were found to be 40 min. (C), 20 min. (0), and 60 min. (C+D). At greater generation times (T), C+D equalled T where C equalled 2/3T and D equalled 1/31. The doubling in cell volume between successive divisions occurred entirely by a doubling in cell length (A) without detectable change in cell diameter (39). E, gglj_divided 20 min. after reaching a particular cell length (2A) independent of the growth rate of the cells (41). Control of cell division by initiation of chromosome replication has been proposed (39, 74, 75, 100). Inhibition of DNA replication by chemical agents, thymine starvation, or ultraviolet (UV) irradi- ation prior to termination completely block cell division, sug- gesting a link between the termination event and the ability of the cell to divide (23, 26, 27, 77, 149). Similarly E, gglj_with mutations in the gggA locus which immediately shut off DNA repli- cation at the restrictive temperature failed to undergo cell division (198). Mutations which uncouple DNA synthesis from cell division have been isolated (87, 103, 155, 159, 200, 214). The uncoupling of cell division from DNA replication resulted in cells with less than a full complement of DNA (89, 94). That one cellular event is responsible for the relationship between replication and divi- sion appears unlikely, since models based on these singular events fail to account for other manifestations. It is possible that a complex sequence of events including both initiation and termination 11 of chromosome replication is involved in the control of cell divi- sion (104). DNA Repair and Cell Division DNA damage inhibits cell division (18, 19, 53, 61, 66, 88, 96, 152, 223) in 3ggA+ (223) 1§5A+ (89, 139) strains of E, 3911. Synthesis of the [EEA gene product, protein X (46, 63, 117, 126), was induced by DNA damaging treatment (95), or at the restrictive temperature in the temperature sensitive (ts) cell division mutant Ejj}l (61, 65, 66). The distribution of protein x was found to be 90% in the cytoplasm and 10% in the membrane. Genetic studies with 125A mutants suggest that the 1255 gene product is a repressor of the gggA gene which is inactivated in response to DNA damage, thus leading to enhanced expression of the [ggA gene (19, 63). Certain types of DNA repair including error-prone, inducible repair were shown to require functional products from the gggA and 135A genes (223). The E, gglj_31f_mutation, located at 51 minutes on the genetic map, is closely linked to the rggA gene (19). At the restrictive temperature, these mutants formed long multinucleated filaments and possessed enhanced levels of DNA repair activity. The gif_mutation was found to be gj§_dominant in partial diploid cells suggesting that the mutation was located in a regulatory region of the gggA gene. Satta and Pardee (180) proposed that protein X provided the connection between damage to DNA, inhibition of septation and 12 obstruction of cell division in E, coli. They examined the ability of several chemical agents, or of growth at the restrictive tem- perature by its division mutants, to inhibit cell division in the presence of rifampin. The concentration of rifampin employed reduced total protein synthesis by 25% and strongly inhibited protein X synthesis. Without rifampin, DNA damaging treatment or growth at 42C resulted in induction of protein X and inhibition of cell division. Cells with chemically or physically altered DNA metabolism divided when rifampin inhibited protein X synthesis. It was concluded that protein X, produced as a consequence of DNA damage, was an inhibitor of septation. Other models proposed to account for the coordination of DNA repair and cell division ascribe an indirect role for protein X and leave open the possi- bility that some other agent is responsible for inhibiting cell division (53, 65, 223). The Cell Envelope as the Site of Coordination of DNA Metabolism and Cell Division Replication of DNA is linked to the cell division cycle, during which genomes must be segregated and partitioned equally between daughter cells (108). Segregation was proposed to be achieved by attachment of the replicating chromosome to the cell membrane (74, 75). Jacob et a1. (97) presented a replicon model for coordination of replication and division which was based on chromosome-membrane attachment; DNA synthesis was correlated with cell growth through signals transmitted to the membrane by the 13 enzyme system of replication. Separation of daughter chromosomes was thought to result from growth of the membrane in localized areas between sites of attachment of the chromosomes. Isolation of membrane attached DNA has been reported in many studies (105, 111, 150, 226). Worcel and Burgi (226) found that DNA was released from the membrane when initiation of DNA replication was prevented by amino acid starvation. Addition of amino acids restored both DNA-membrane attachment and DNA repli- cation. Isolated DNA-membrane complexes contained a variety of inner and outer membrane proteins (150). Cross-linking of bromodeoxyuridine-substituted DNA with membrane proteins, induced by UV irradiation, permitted the isolation of two proteins in close association with DNA. A major role for these 80 Kilodalton (KD) and 56 KD proteins of the inner membrane, in DNA-membrane attachment, was proposed. The origin and replication point of the E, gplj_chromosome (146) and DNA, enriched in genetic markers located near the origin of the Bacillus subtilis genome (203), sedimented with membrane in sucrose gradients. The association of these DNA sites with cell membrane was a necessary assumption of the replicon model. Studies on cell wall growth were used to derive evidence for localized membrane growth based on the assumption that their growth pattern was similar (200). Using 3H-diamminopimelic acid (DAP) in pulse and pulse chase experiments, Rhyter et al. (179) demonstrated that murein was deposited in the cell wall at central sites near the presumed septation site in E, coli. The incorporated 14 3H-DAP appeared to spread randomly over the entire cell surface after a short chase period. Murein synthesis was greatest in cells which were ready to divide (3). Treatment of E, ppli_cells with low concentrations of penicillin produced characteristic equatorial bulges as a result of weakened areas of the cell wall (17, 40, 182). The position of these bulges was hypothesized to correspond to sites of murein synthesis where normal cell division occurred in the absence of the antibiotic. Modifications in the cell envelope protein composition resulting from alterations in DNA metabolism have been reported (23, 62, 66, 95, 99, 180, 195). Under normal growth conditions, a 76 Kd protein, synthesized during a brief period near the time of cell division, was inserted into the membrane within four minutes of its synthesis, and quickly lost in the succeeding generation (23). An 80 Kd outer membrane protein was shown to be synthesized at the time of initiation of DNA replication (62). James (98) reported induced synthesis of an outer membrane lipoprotein (protein G) when DNA synthesis was inhibited. Specific inhibition of septum formation had no effect on its synthesis suggesting that protein G was solely involved in events that occur when DNA synthesis was blocked. Inhibition of DNA replication in gpr mutants resulted in a deficiency in a 60 Kd membrane protein coupled to a quantitative increase in a 30 Kd protein (195). A decrease in a 35 Kd protein with a concomitant increase in a 25 Kd protein was observed in membranes of gpgA mutants grown at the restrictive temperature. Ricard and Hirota (159) failed to detect alterations in membrane 15 composition in f3§_mutants which failed to divide at 42C. The EIEA gene product has recently been identified as a 50 Kd protein (119) which is required throughout the septation process. Inouye and Guthrie (95) observed that E, gplj_protein Y decreased and protein X increased in membranes of temperatures sensitive DNA replication mutants grown at 42C. In subsequent work (61, 65, 66, 96) this alteration was noted in cells where DNA synthesis was chemically blocked, and in its cell division mutants Elf (18) and E§l_(139), which replicate DNA at the restrictive temperature. A system of control of DNA repair and cell division comprised of the 135, gggA and Ejf_genes was pro- posed to account for these observations (61, 65). Constituitive DNA Repair Processes Certain types of cancers appear to result from cells deficient in DNA repair processes. For example, sunlight induces skin cancers in repair-deficient cells of humans with an heredi- tary disease known as xeroderma pigmentosum. A number of tumors differ in their sensitivity to platinum(II) compounds, possibly due to their DNA repair capabilities. Examination of DNA repair mechanisms remains difficult in mammalian systems due to the complex structure of chromatin and the scarcity of mutations in DNA repair processes. The possibility also exists that cultured cells have altered DNA repair activity (68). Determination of the mode of action of platinum(II) compounds is facilitated in E, coli cells because of the availability of a large number of 16 mutants deficient in different DNA repair processes, and, the characterization of some of the gene products involved. DNA repair in bacteria has been extensively reviewed (59, 67-70). Photoreactivation, excision and recombination repair are three constituitive repair processes which function to maintain the integrity of DNA in E, ggli_(67). Pyrimidine dimers induced by UV-irradiation are the most commonly studied DNA 1esions. They consist of adjacent pyrimidine bases, cross-linked through their respective 5 and 6 carbon atoms to form a cyclobutane ring. Dimers distort the secondary helical structure of DNA causing local denatu- ration due to the inability of affected bases to hydrogen bond with bases in the complementary strand (189). Photoreactivation Pyrimidine dimers were reverted to their constituitive pyrimidines by a process which involved photoreactivating enzyme (PRE) and photoreactivating light (PRL) (175, 188). Monomerization of dimers by PRE required light with wave lengths ranging from 300 to 500 nm. (189). The chemical mechanism of this event has not been determined. PRE, which binds specifically to pyrimidine dimers on single strand regions of DNA (176, 187), was identified as the product of the pppA and pppB loci (203). Excision Repair Incision of DNA strands containing adducts, excision of the adduct, repolymerization and ligation of the repair patches comprise the set of coordinated activities known as excision repair (8, 43, 17 59, 60). Differences in sensitivity to DNA damaging agents led to the isolation of mutants deficient in various stages of this process (37, 52, 85, 141, 217). Mutants, in the pyrA, 33:8 and EXEC loci, isolated on the basis of their increased sensitivity to UV light and alkylating agents but not to agents causing strand scission, were deficient in excision of pyrimidine dimers and other DNA damage (85, 106, 201). The 315A and 3358 gene products corendonuclease II (59) was characterized as an endonuclease which bound specifically to UV irradiated DNA (9). The endonuclease activity incised DNA at the 5' side of dimers, or one nucleotide removed, resulting in the formation of a 3' hydroxyl terminus thus providing a site for initiation of polymerization (58). Caffeine, which binds to dimers (49), competitively inhibits the activity of this enzyme (10). Studies on toluene treated cells and jp_ij§9_complementa- tion assays indicated that the Eli? gene product prevented ligation of incised DNA (183, 191). The physiological significance of p150, 213E and gng loci have not been determined (68). Radman (153) isolated a second UV-irradiated DNA specific endonuclease activity, endonuclease ILL from cells deficient in corendonuclease II. The 5' to 3' exonuclease activity of DNA polymerase I was shown to be responsible for the excision of a majority of dimers f61lowing incision by the pyrA 2358 gene product (20, 21, 79). Exonuclease V, exonuclease VII and the 5' to 3' exonuclease asso- ciated with DNA polymerase III exhibited dimer excision but their activity appeared to be limited (22, 33, 43, 192, 227). 18 DNA polymerases I, II and 111, products of the ppLA,.png and gng genes respectively, are all capable of repair polymeri- zation under certain circumstances (108, 125, 206). Cooper and Hanawalt (34) provided evidence for two gap sites resulting from dimer excision; short gaps of approximately 30 nucleotides, and long gaps of approximately 1500 nucleotides appeared to be repaired by different mechanisms. Repair of short gaps by DNA polymerase I and long gaps by DNA polymerase III was demonstrated (2, 33, 48, 57). Exonuclease V was implicated in the formation of long gaps (33). Grossman et a1. (59) presented models for the repair of both types of gaps. DNA ligase (56, 143, 228, 230) is required to rejoin the repair patch to the phosphodiester back bone of DNA. Temperature sensitive ligase mutants were radiation sensitive, and formed filaments at the restrictive temperature (52). Recent evidence suggested that DNA ligase exerts some control on DNA polymerase I repair of X-irradiated DNA (6). DNA glycosylases which cleaved the glycosidic bond between deoxyribose and purine or pyrimidine bases have been isolated from E, 9911 cells. Uracil, hypoxanthine and 3-methyladenine DNA glycosylase activities were identified but the importance of their function in DNA repair remains obscure (113). Recombination Repair Extreme UV sensitivity and an almost total deficiency in recombination ability of certain E, coli isolates resulted from a 19 mutation in a gene designated as pepA (24, 25, 88). The product of this gene was shown to promote genetic recombination, DNA renatur- ation, and homologous pairing between single strand and superhelical DNAs (82, 190, 216). The pleiotropic responses under control of the [egA gene is discussed in terms of the SOS hypothesis. Mutations which mapped in separate genes distinct from the gegA region, pegB and :egC, imparted a less severe reduction in recombination activity to E, gpli_cells (45, 217). These genes were found to code for subunits of exonuclease V (24), an enzyme with an ATP-dependent 5' to 3' and 3' to 5' exonuclease activity which hydrolyzes duplex or single strand DNA to oligonucleotides, and a single strand specific endonuclease activity (114). In the presence of a DNA binding protein, exonuclease V acted as an ATP- dependent DNA unwinding enzyme, able to generate large single strand DNA regions (170). The :egA protein has been shown to bind specifically to single strand DNA (66), and may act jp_!i!g to promote the unwinding activity of exonuclease V in a [egA, gegB, £229 dependent recombination pathway. The phenotype of gegB' and regC' derivatives is suppressed by mutations designated as gpgA and gpr. The §pgA mutation was recessive in merodiploids (118); strains with regBC epgA genotype were found to have a new ATP independent exonuclease activity, exonuclease VIII (109). A muta- tion in the gpr locus resulted in a deficiency of exonuclease I (110), an enzyme which degrades single strand DNA from 3'0H termini. A mutation in either gene resulted in UV resistance and recombina- tion proficiency in recB or recC mutants. 20 A gegA dependent, [egBC independent recombination pathway was demonstrated in recB recC sch mutants. The activity of this RecF pathway was implicated in certain types of recombination which the RecBC pathway cannot perform (174). The legA mutation (eEEA in E, coli B), was shown to result in loss of some recombi- nation ability (138) and loss of inducibility of SOS functions (223). The 121A gene product was recently isolated (116) which exhibited DNA binding properties (0. W. Mount, personal communi- cation). Ganeson and Seawell (50) hypothesized that the activity of the legA and Eegf genes were required directly or indirectly in excision deficient cells for the joining of short DNA segments into longer strands, concomitant with the transfer of DNA from irradiated templates into unirradiated daughter strands. The replacement of long stretches of single strand DNA by the cor- responding strand from donor DNA in a non-reciprocal manner was thought to be mediated by the RecF pathway (122). Chromosome breakage was observed during genetic recombina- tion (131). Rupp and Howard-Flanders (177) found that postrepli- cation repair involved a mechanism distinct from the excision repair pathway. Using density and radioactive labeled DNA, they demon- strated the exchange of DNA between sister duplexes (178). Super- infection by phage whose DNA contained interstrand cross-links promoted genetic recombination in py§A+ gegA+ host cells (86). Scission of circularized A DNA was observed in E, gplj_which were superinfected with cross-linked A DNA. This "cutting in Eggpe? occurred only between homologous DNA molecules in a gegA+ host 21 cell (83). Mismatch repair, an excision repair function which acts on mismatched bases in heteroduplex DNA (151), was implicated in the process of genetic recombination (130). It was postulated that a difference in methylation of DNA permits excision of mis- matched bases to be directed so that the parental strand is con- served and the unmethylated strand is repaired (55, 213). Glickman et a1. (55) isolated mutants deficient in methylation, ggm72 and ggmy3, which were mutated by base analogues at much higher rates than the parental strain. A role for DNA methylation in error avoidance during DNA replication was suggested. A model for genetic recombination was recently proposed by Radding (151). Salient features of this model included strand displacement, uptake of the displaced strand by a homologous duplex, cleavage of the 0 loop produced by strand uptake, assimi- lation of the displaced strand by 5' to 3' exonucleolytic digestion, isomerization resulting in two strand crossover, and branch migra- tion, resulting in symmetrical exchange of strands and symmetrical formation of heteroduplex DNA. Repair of Interstrand Cross-Linked DNA Repair of interstrand cross-links was shown to be dependent on both excision and recombination repair processes (30). Repair occurred sequentially by incision of DNA containing cross-links by the 335A,.g!§B gene product (31) and gap filling by a recombination pathway requiring functional products from the regA, 3e98, :egC, §Q§o reef and 1258 genes (196). Some DNA synthesis was required, 22 presumably for strand displacement in the formation of recombinant molecules. The SOS Hypothesis As a result of certain alterations in DNA metabolism, E, gplj_exhibit a number of new activities such as prophage induc- tion, filamentous growth, mutagenesis, and increased DNA repair capacity whose expression appears to be coordinately controlled (221). These responses have been grouped into a general model termed the SOS hypothesis because they are thought to aid in cell survival under conditions where DNA is damaged (152). The expres- sion of these processes depends on a le5A+, regA+ genotype. SOS functions are expressed in Eif_mutants at the restrictive temper— ature. Work with this mutant suggested a common regulation pathway for these activities (18). Induction of SOS Functions Weigle (215) found that survival of UV-irradiated phage X increase if host cells were irradiated prior to infection. This survival (UV-reactivation) was accompanied by a high level of phage mutagenesis (UV-mutagenesis) suggesting that some repair enzymes were induced in the host by UV irradiation (12, 36, 107). UV reactivation and mutagenesis were maximally expressed 30 minutes after inducing treatment (35). The presence of chloramphenicol (CAM) in cultures, for 40 minutes after irradiation, completely blocked these activities. CAM added prior to UV irradiation inhibited a small amount of postreplication repair and prevented 23 mutation fixation in excision-deficient (pygA’) mutants of E, £911, (184). When protein synthesis occurred between inducing and CAM treatments, repair was completed and mutations were fixed. When EjjlpypA mutants were incubated at 42C prior to irradiation, repair and mutagenesis occurred in the presence of CAM. The conclusion of these studies was that error-prone DNA repair, manifested under DNA damaging conditions, was an inducible activity. DNA Damage Reeponsible for SOS Induction The role of DNA damage in $05 induction was confirmed by George et al. (54) who demonstrated that F' recipients of E, 9911, that received an F+ or Hfr plasmid DNA from an irradiated donor, exhibited UV reactivation and mutagenesis when subsequently infected with a UV-irradiated phage X. Daughter strand gaps resulting from the excision of or replication over non coding pyrimidine dimers were postulated as inducing signals for SOS functions (224). Boiteux et a1. (7) suggested that the 3' to 5' exonuclease activity of DNA polymerases, acting at these gaps, produced deoxyribonucleoside monophosphates which act as the inducing signal. Inducing treatments which directly caused DNA strand scission (i.e., bleomycin, colicin E2) resulted in a more rapid induction of SOS activity than those which indirectly resulted in gaps (i.e., UV irradiation) (199). DNA degradation was required for inactivation of X repressor; a mutation in the gegB gene delayed inactivation, implicating the gegBC exonuclease 24 in the induction pathway (142). No induction of SOS activities was observed in [e28 :egC mutants when DNA replication was inhibited by naldixic acid (61, 65, 66). In contrast, Little and Hanawalt (115) observed UV induction of SOS functions in a gpgAlgegB mutant grown at the restrictive temperature. Under these conditions, naldixic acid treatment had no effect. It was concluded that the gepBC exonuclease was not absolutely required for induction. Target for Error—Prone Inducible Repair Activity Sedgwick (185, 186) obtained evidence that overlapping daughter strand gaps, on opposite strands of the same DNA molecule or on the same strand of different molecules were refractory to constituitive excision and recombinational repair processes. These lesions were proposed as a target for SOS repair activity. Witkin (223) employed this hypothesis to explain the dose-squared muta- tional effect: at low doses of UV light, the mutation frequency increases as the square of the dose. This model assumed that both single strand and overlapping gaps were capable of inducing and being acted upon by SOS repair processes. It was suggested that at UV doses resulting in suboptimal SOS induction, UV induced mutagenesis required one photon absorption (hit) in a specific gene and an SOS inducing hit somewhere on the chromosome. At UV doses where SOS repair was maximally induced, a second hit in a gene proximal to a previous hit in the opposite strand would result in a lesion capable of being repaired only by error-prone repair and 25 the dose-squared effect would continue. A dose-squared relationship was shown to exist until the maximum mutation frequency was reached (224). Enzymatic Activities of SOS Repair Demonstration of inducible error-prone repair in pplA (222, 224) pygA (184, 185), pepB‘pegC (80, 134, 220) and regf (223) mutants of E, £911 ruled out mechanisms of DNA repair involving the products of these genes. Bridges and Mottershead (13) found that slowly growing cells with largely unduplicated genomes exhib- ited no significant difference in mutability from fast growing cells possessing duplicate chromosomes. This result implied that pegA dependent mutagenesis was not the result of genetic recombination between homologous chromosomes. Fidelity of constituitive DNA polymerases to template instruction (108) appeared to rule out their involvement in SOS repair. Bridges et a1. (16) found that UV- irradiated pypA pplC double mutants fixed mutations at 34C but lose this activity when shifted to the restrictive temperature. DNA synthesis continued for a short period after the temperature shift, but there was an immediate loss of mutation fixing ability. An activity of DNA polymerase III was suggested to be necessary for SOS repair. Subsequently a pplC revertant was isolated which was deficient in SOS repair at 340, indicating the involvement of DNA polymerase III in inducible error-prone repair (11, 14, 15). Radman and co-workers (7, 154, 211, 212) demonstrated that E, coli DNY polymerases I and III, which possessed a 3' to 5' exonuclease 26 (proof-reading) activity, did not incorporate deoxyribonucleoside triphosphates (dNTP) into DNA opposite noncoding lesions in the DNA template. They observed a high rate of turnover of dNTPs to dNMPs caused by the proof-reading exonuclease. Certain mammalian poly- merases, free of this activity, polymerized through noncoding 1esions. It was concluded that SOS repair polymerization was due to inhibition of the 3' to 5' exonuclease function by some inducible agent. In contrast, different metal activators in jp_vitro E, coli DNA polymerase I assays increased the error rate of polymerization up to 15 fold without significantly altering the 3' to 5' exo- nuclease activity. Misincorporation of dNTPs could result from an effect other than loss of polymerase associated proof-reading activity (197). Genetics and Regulation of SOS Functions The pleiotropic effects shown to result from mutations in the E, pplj_pegA gene included a deficiency in genetic recombina- tion (25), sensitivity to UV-irradiation (25, 88), inability to induce prophate X (38, 78), absence of mutability (219), inability to coordinate DNA synthesis with cell division (94) and spontaneous extensive degradation of DNA (24). The pepA gene product was iden- tified as protein X (46, 63, 126, 127, 128). Its observed activi- ties were binding to single strand (55) DNA, an ssDNA dependent ATPase activity, promotion of ssDNA renaturation (216), facilitation of recombination (190), and cleavage of the 1258 gene product (D. W. Mount, personal communication) and of the phage X repressor in an 27 ATP dependent reaction (164, 165). The Elf mutation which is thermoinducible for all SOS functions was mapped in the regA gene (18). Cells possessing the Eif_mutation produce an altered form of protein X (46, 63, 126). Mutations in the 1215 gene also mapped in the pegA region (136). These mutants were distinguished from [egA' strains because they were less sensitive to UV and X irradia- tion, showed wild type spontaneous DNA degradation properties, were able to perform some genetic recombination, and exhibited some UV— reactivation. Mutations in the gfi locus were isolated for their ability to specifically suppress filamentous growth in 31: and Ejjllpp_ mutants (53). Witkin and Kirschmeier (225) proposed that §jj_ mutations participate in SOS regulation via effects on the activity or specificity of cellular proteases. A mutation in the le5A gene resulted in increased sensi- tivity to UV and ionizing radiation (84) and decreased mutability (218), but genetic recombination was not drastically affected by this mutation. This gene mapped at a different site than the gegA locus (19). The legA gene product, recently identified as a 24 Kd protein (116), had DNA binding properties, and is cleaved by the gegA gene product jp_yjpgp_(0. W. Mount, personal communication). Genetic studies suggested that the 1e5_gene product is a repressor which regulates the expression of the pegA gene (19). The 1e§A mutant form of this repressor molecule has been characterized as a superrepressor, which is resistant to proteolytic cleavage (65). The t§l_mutation suppressed the radiation sensitivity (51) and 28 increased the postreplication repair capacity of lexA mutants. This thermosensitive mutation was found to be closely linked to the 125A locus (139). Similarly, the §p§_mutation (137), an amber mutation in the 1218 gene (154) caused constituitive expression of lysogenic induction and error-prone repair. Models proposed for the regulation of expression of SOS formations (46, 47, 63, 65, 126, 223), incorporated the 125A gene product in a repressor role, the protease activity of the [egA gene product, and DNA degradation products as a signal for induc- tion. The Ejj_mutation was located in a regulating region of the regA gene, although recent evidence that protein X from Elf mutants differs from the normal reg_A gene product, indicates that it may be in a structural gene. A representative model, proposed by Gudas and Mount (63) is shown in Figure 1. Protease Activity and SOS Regulation A protease activity was implicated in the induction of SOS functions by Roberts et al. (164, 165), who found that thermal induction of prophage X in El: mutants resulted in cleavage of the X repressor. Identical cleavage products were produced when this repressor was acted upon by the [egA gene product lg giggg. Meyn et al. (133) observed that a competitive protease inhibitor, anti- pain [(1-carboxy1—2-pheny1ethy1)carbamoyl-L-arginy1-L-va1ylargininal], prevented X induction, UV mutagenesis and filamentous growth in_§i[ mutants. Introduction of a plasmid carrying the X repressor gene (cI) into Eli §f1_mutants of E, coli decreased the induced mutation 29 frequency by 50% (132). It was suggested that the repressor, con- trolling expression of SOS dependent mutagenesis (error-prone repair), possessed a cleavage site similar to that of the A repressor. Tosyl-leucyl-chloromethyl-ketone, an irreversible protease inhibitor, blocked the expression of SOS functions (154). Swenson and Schenly (204) observed that low doses of antipain did not significantly effect respiration, viability, or growth of E, 9911 cells. Administration of this compound to UV-irradiated cells significantly decreased these cellular activities but the effect was transient. Antipain treatment had no effect on exci- sion of pyrimidine dimers. 30 Figure l. A model for the role of 99£A protein X in the induc- tion of SOS functions. During normal growth, the product of the 195A gene represses the synthesis of the 99£A protein. The 999A protein recognizes DNA damage, perhaps by the accumulation of a low molecu- lar weight inducer molecule which interacts with this protein. Activated protein X then destroys the 195A repressor by proteolytic cleavage, derepressing its own synthesis. The 111 mutation alters the damage recognition site of protein X resulting in spontaneous activation in the absence of alterations in DNA metabolism. The 999_mutation lowers the bind- ing affinity of the repressor resulting in constitui- tive protein X synthesis. Damage recognition and autoregulatory properties of protein X (127, 129, 145), allow for a high level of this protein in its biochemically active form for the induction of various SOS functions (63). 31 Aommmoficdv 8 £29m umfi>zu< 88 w 88:86:23 x VOW IV% 8039.. xx 00 H commocaom flaw VA I|...< , a :UT x4 “ a + 838 <20 «I. Co «E 10. 32 Literature Cited Anderson, K. S. 1979. Platinum(II) complexes generate frame shift mutations in tester strains of Salmonella typhimurium. Mutat. Res. 91; 209-214. Barfknecht, T. R., and K. C. Smith. 1978. The involvement of DNA polymerase I in the post replication repair of radiation-induced damage in Escherichia coli K-12. M01. Gen. Genet. 191, 37-41. Beck, 8. D., and J. T. Park. 1976. Activity of three murein hydrolases during the cell division cycle of Escherichia coli K-12 as measured in toluene-treated cells. acteriol. 1E9: 1250-1260. 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Clinical evaluation of the toxic effects of gig-diamminedichloroplatinum (NSC- 119875)-phase I clinical study. Cancer Chemotherapy Repts. 61; 465-471. Tait, R. C., A. L. Harris, and D. W. Smith. 1974. DNA repair in Escherichia coli mutants defective in DNA polymerases I, 11, and/or III. Proc. Natl. Acad. Sci. U.S.A. 11; 675-679. Taylor, R. T., J. H. Carver, M. L. Hanna, and D. L. Wondres. 1979. Platinum-induced mutations to 8-azaguanine resistance in Chinese hamster ovary cells. Mutat. Res. 61; 65-80. Vanden Berg, H. W., and J. J. Roberts. 1975. Alkaline sucrose gradient studies: changes in the sedimentation profile of template DNA from Chinese hamster V9-379A cells treated with the antitumor agent 616-p1atinum(II)- diamminedichloride. Studia Biophys. 66; 39-46. Vanden Berg, H. W., and J. J. Roberts. 1975. 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The involvement of polynucleotide ligase in the repair of UV-induced DNA damage in Escherichia coli K-12 cells. Mol. Gen. Genet. 152: 37-41. Zak, M., J. Drobnik, and Z. Rezny. 1972. The effect of 612-platinum(11)-diamminodichloride on bone marrow. Cancer Res. 62; 595-599. Zimmerman, S. B., and C. K. Oshinsky. 1969. Enzymatic join- ing of deoxyribonucleic acid. III. Further purification of the deoxyribonucleic acid ligase from Escherichia coli and multiple forms of the purified enzyme. J. Biol. Chem. 244; 4689-4695. SECTION II 54 Fragmentation ot eta-platinum(II) d’ammlnodtchlorida-inducad filaments oi wild type and DNA repair-deficient mutants of Escherichia coli. Bruce E. Manama a Robert R. Bnrunn. Department 11 .tlicrnI-iotngy and Public Health. Michigan State i'niversitg. East Lansing. Michigan 4882‘ Introduction. The anti— tumor compound cis- ~pIatinnmtIl1diammi- nodiehloride (PhD) is :1 potent radii-mimetic bacterial mutagen .l \1hich induces filamentous gmlh of 111111 ture and certain 1I1na1ritmnucleic acld IDNAI repair- -11cti1icnt mutants of Escherichia roll '2.) PDD 1an re1ersllil_v Inhibit s1nthesIs at l.\'.«\ III E. coli _2 and induce temperate phage in I1 sogenic bacteria 3 . In this report “e compare frequencies of PDD induced filamentation and times required for subse- quent fragmtntation alter rcmo1al oi the compound. Itesults suggest that mutations that promote long term maintenance of single stranded regions of DNA t1.e. recA, rccltC, and Hunt) are unable to recover abi- lity to divide after exposure to PDD. Methods and Materials. Medium. Defined medium :41 supplemented uith o—glucose (10 m.\l). L- arginine. L-tr_1ptophan. L- Icucine, L- threonine, L- histidine. L- proline. L-methionine (all l.0 111.“). thiamine (5 pg g'ml) and thymine (50 pg/ml) “as used in all experiments. In some cases this me- dium “as solidified with 15 per t Bacteria. .-\II .A repair-deficient isolates mere obtained from Dr. B. Bachman except strains D\ 145 and DY 194 11 hich \sere received trom Dr. D. \‘oungs. Filumentalion. Exponentially growing cells were hnnested and resuspcnded at an optical density of 0.0.3 (6'20 11111) in medium (10 ml/l25 ml Erlenmeyer ilasln containing the 1011' concentration of PDD (0.5 pg.’ml). Atter aeration at 37' (unless otherwise speci- fied) for I'.’ h in a model 676 gyrotory water—hath shaker (New Brunswick Scientific 00.). sam Ies were removed and diluted in gl_1*cerol:tormalin counting solution. Frequenev ot filamentation nas determined bv observation by phase contrast microscopy using a Petroft- Hauser counting chambc c.r Cells recorded as filaments exhibited a length equal or greater than :1 times that of Icontrol organisms. I Fragmentation. Uells \sere culti1ated as previouslg described for 4 h 111th the high concentration of PD D (5.0 ug/mll. \sashed tuice “1th cold 0.033 .\I potas- sium phosphate buffer. pH 7.0. and then pla edon the surface at agar slide cultures (PDD-free solid defined medium). The cultures were observed at intervals and times were recorded when the filaments underwent bagmentation. “reagents. PDD was generously provided by Dr. D. h. Results. Filamentotion. D.\'A repair-deficient mutants block- ed in either excision or recombinat on repair were examined for ability to undergo filamentation at the low dose (table ll or high dose (table II) of PDD. Results obtained with isogenic prototrophs were simi- lar ; thus only data obscr1ed tor strain AB "57 (wild type) is presented. Wild t1- rpe. point and recB.C Iso- lates tormed filaments at the high dose hut cw normaII_1 at the low concentration. In contrast. the highd use “as toxic for tea and especialls recA cells “hieh “ere killed before filamentation could occur. Filamentation oi uvrA isolates occured at both con- centrations of PDD and tigta cells undenent stapon neous filamentation “hich “as enhanced by addition PDD (1011' dose) at permissise temperatures Fragmentation. Cell division of stashed ‘Ild tspe and pol.4 III aments commenced < 25 and 15 tare tivelv. In contrast. filaments at rectl.C and norA mn- tant's continued to elongate tor 24 h aite r removal of PD!) ulthout undergoing tragmentation (table II). Slmilarh. filaments or recA cells induced at the ow dose tailed to divide by hnhercas those of le: Isolates commenced dIvIsIon to h after removal of the compound Tuna I. ' PDD induces filamentation in DNA repair—deficient mutants of E. coli. Percentage oi population Ilamented (I) face“ Straia Altered gene “an” ‘ 'M'“ ' with PD!) ' film,“ pop “15 KIWI llamntatioa AB 1157 117’ None 0.3 I t 0.! AB 2463 recA Protein X 1.0 46 5 45.5 JC 5519 reeBC Exonuclease V 0.0 I 6 1.0 MM 383 1191.4 . DNA polymerase I 0.5 I 3 0.0 AB 1586 nrrA Correndonuelease II 0.6 9.0 0.4 DY 145 uan_ Correndonuclease II 2.6 It] 10 5 AB 2494 lead Unknown 0.0 4 0 4.0 A8 2474 and tad —- 0. 3 11.8 “.5 DY 191 119117 DNA ligase 28.1(c) 38.8(c) 111.7 DY 194 “9137 DNA ligase 45 0th) 54.0(b) 0.0 (111 approximatcls 5 to I0 times longer than 1ontrol cells 11' (b) grout: at the restric- the temperature 42': 1c) )gronn at the permissi1e tempera 55 56 Tan: 11. Fragmentation of PDD induced filaments. I'DD i‘ragmeu- ‘I’itne induced lation alter olinitiation bu." filaments removal oi lrsgnun- (5 ugltnl) ol PDD lation (11) AB 1157 111‘ + . + < 2.5 A8 2403 recA . 0 AB 2470 recB + 0 JC 5519 rech + 0 _ . MM 383 1101.4 . + + 15 AB 1886 uvrA + 0 ES 2 uvrA 1101.4 + 0' AB 2404 test 0 AB 2474 uvrA 1d 0 ° Discussion. Due to uncontrolled exonuclease activity. both recB.C, lea and especially rcc.4 isolates are unable to efficiently close gaps (armed in their DNA processes of excision or non-reciprocal combination ;5. 6,. Although tea and recA cells undec11ent filamentation at the low dose of PDD. subsequent fragmentation «as not detected with recA. Analogous results were obtained for recB,¢ cells at the high dose 01 PDD. Similarly. cells blocked in excision (uvrA) failed to initiate division after removal of exogenous PDD. Un- excired PDD-nucleotide complexes in these organisms would result in formation of gaps during subsequent replication. In contrast, filaments of pot.l mutants underwent delayed fragmentation due. presumably. to compensatory activity of the dnu£ product L7). These results are consistent with the hypothesis that the presence of gapped DNA' (which cannot un- dergo supercoilingl prevents initiation of cell divi- sion. Whenever cell division remained inhibited after removal 01 PDD. the organisms possessed mutations 11 hich delayed ligation (tea. recA. recti.C) or assured the occurcnce of gaps in ne1viy replicated DNA (until. Furthermore, the tigls mutant underuent extensive spontaneous filamentation. This observation uould be expected it gaps in DXA were indeed responsible for inhibition of cell division. . Bedl'B 101 8.19. a Brubaker. R. R. (1975) Slut. Res” 27. . Beck. D. J. a Brubaker. it. It. (1973) 1. Bacteriol” 115. 1247-1252. . Iteslova, 5. (1971/72) Chem. Biol. lnterocl.. 4. 56-70. . Neidhardt. F. C., Bloch. P. 1.. a Smith. D. F. 119711 J. ”acteriol.. 119. 736-717. . Clark. A. J. (1973) Annu. Rev. Genet. 7. 57-86. . Mount. 1). “2, Low, K. B. a Edmistom S. J. (1972) J. Bacteriol. 112. 886-893. . Youngs, D. .4. a Smith. R. C. (1273‘ Nature New Btol.. 214. 210-241. ban-l st ’9' A SECTION III 57 Influence of Chromosome Integrity on Cell Division in gj§:Platinum(II)diamminodichloride-Induced Filaments of Wild Type and Deoxyribonucleic Acid-Repair Deficient Mutants of Escherichia coli Bruce E. Markham* and Robert R. Brubaker Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 *Present address: Department of Microbiology College of Medicine University of Arizona Tucson, AZ 85724 58 A low dose (5 ug/ml) of the antitumor agent gjsyplatinum(II)- diamminodichloride (PDD) caused wild type and deoxyribonucleic acid (DNA) repair-deficient mutant cells of Escherichia coli K-l2 to grow as long multinucleated filaments. At this concentration, the times required for reduction of viability to e'1 for wild type, 9915, rggfilg, and uvrA_organisms was >200, 200, l20, and 25 min, respec- tively. As shown by sedimentation in alkaline sucrose gradients, generation of single strand breaks in DNA of these organisms was a major consequence of growth in PDD. Upon incubation in fresh medium after removal of PDD, a respective lag of l, 2, and 4 h occurred before filaments of wild type, 291A, and Egg§,§_cells commenced cell division. This procedure failed to promote similar fragmentation of gyrA filaments unless the organisms were maintained at 4°C for at least 4 h. After such storage, which appeared to delay subsequent initiation of new rounds of chromosome replication, a lag of 3, 4, 6, and 9 h was observed after transfer to fresh medium and the onset of fragmentation in wild type, polA, recB,C, and uvrA filaments, respectively. In all cases, these periods of time corresponded to those required for restoration of normal chromosomal molecular weight in alkaline sucrose gradients. These results indicate that ligation of PDD-induced gaps in DNA is requisite for the occurrence of cell division and are in accord with the hypothesis that the presence of such gaps promotes expression of an inhibitor of cell division. 59 ....§.. out .uuu .‘IIII 60 Although first described as an antitumor agent (30), gjs: platinum(II)-diamminodichloride (PDD) was later found to be muta- genic in tissue cultures (36) and carcinogenic in mice and rats (16). Antitumor activity possibly reflects the ability of P00 to inhibit net synthesis of deoxyribonucleic acid (DNA) in eucaryotic cells by irreversible generation of interstrand cross-links (13, 14, 2l, 27). However, the compound did not significantly inhibit rates of DNA synthesis in Escherichia coli (4), an observation in accord with the known capability of this organism to remove inter- strand cross-links (9). Nevertheless, as in the case of mammalian cells, PDD was radiomimetic in E, coli (5, 8, 25, 29, 33) and related procaryote species (15). This finding is consistent with recent observations indicating that formation of intrastrand cross- links between adjacent guanine residues is a major effect of PDD on DNA (8, 28, 34). These lesions can probably be eliminated by estab- lished mechanisms of excision repair (4, 19) thereby accounting for the mutagenic and thus carcinogenic potential of the compound (12, 37). When maintained in the presence of PDD, cells of g, 9911 grew in the form of very long (>50 um) multinucleated filaments (29, 34) that contained significant levels of rggA_product [protein X (l7)] (D. J. Beck, personal communication). These pleiotropic responses to a radiomimetic agent are characteristic of E, gglj_ expressing SOS functions (22, 38). This phenotypic state evidently facilitates survival of cells undergoing repair of severely damaged DNA (32) but can also be induced in temperature sensitive 51f, 61 mutants without use of ultra-violet irradiation or radiomimetic agents (5, 6). George et al. (ll) suggested that E, 9911 possesses a control system, sensitive to lesions in DNA, that regulates pro- duction of an inhibitor of cell division. Implicit in this model is the notion that completion of DNA repair precipitates decay of this inhibitor thereby resulting in initiation of septation. In this study we examined the physical state of the E, coli chromosome during growth with P00 and after its removal. The results indicated a temporal relationship between ligation of PDD- induced breaks in DNA of wild type and DNA repair-deficient mutants and the onset of cell division in filaments. This correlation is consistent with the hypothesis (l8) that chromosomes in PDD-induced filaments must return to a normal supercoiled configuration (38) prior to the initiation of cell division. MATERIALS AND METHODS Bacteria Genotypes of isolates used in this study are shown in Table l. Identical results were obtained for parent strains of DNA repair-deficient mutants thus data for only strain W3llO is pre- sented in comparisons of PDD-dependent effects on wild type and mutant organisms. Media and Cultivation Defined morpholinopropane sulfonate-buffered medium (20) supplemented with D-glucose (0.0l M), thymine (2 pg/ml), thiamine (l0 pM) and 0.2 mM each of L-arginine, L-histidine, L-isoleucine, L-valine, L-proline, and L-threonine was used in all experiments; viability was determined on Difco nutrient agar (Detroit, MI). Buffer used for diluent and wash solution was 0.033 M potassium phosphate, pH 7.0 (phosphate buffer). Before use in experiments, bacteria were grown at 37°C for at least eight doublings by aera- tion in a model G76 gyrotary water bath shaker (200 rpm) (New Brunswick Scientific Co., Inc., New Brunswick, NJ). Filamentation Exponentially growing cells were suspended in medium con- taining P00 (5 pg/ml) at an optical density (620 nm) of 0.05 and incubated for four doubling periods (m4 h). The induction of 62 63 filamentous growth was determined by direct microscopic observa- tion. Yields of over 80% filaments were routinely obtained with wild type, 2915, and uvrA_organisms whereas cultures of rggB;§_ cells contained only 50 to 60% filaments. To concentrate filaments of rggBl§_cells, 50 to 100 ml of culture was filtered on no. 1 glass fiber filter paper (Whatman, Inc., Clifton, NJ) and the fila- ments were removed by manual agitation for l min in 10 ml of phos- phate buffer. This procedure produced cell suspensions containing 80% recB,C filaments. Fragmentation of Filaments PDD was removed from filaments by two methods. The first, termed wash by filtration, involved filtration of cultures con- taining PDD with 0.22 pm pore size membrane filters (Millipore Corp., Bedford, MA) following by washing with two culture-volumes of phosphate buffer. The filter was then immersed in 5 ml of phosphate buffer and filaments removed by manual agitation for 1 min. The second method, termed wash by centrifugation, was performed by chilling cultures of filaments for 5 min in an ice- bath, centrifugation at 17,000 x g for 15 min at 4°C, and suspension of the pelleted organism in 1 volume of cold phosphate buffer; these preparations were held at 4°C for at least 4 h. Suspensions of filaments obtained by either method were used to inoculate prewarmed (37°C) medium at an optical density of 0.3. Gram studies were pre- pared after each hour of incubation and the percentage of filaments per microscopic field was determined; at least 3,000 cells were 64 counted per slide. Similar determinations were made using a petroff-hauser counting chamber. Analysis of division on agar slides was performed as previously described (18). Net Synthesis of DNA Cells were grown with [3H]thymine (7 uCi/pmole) for eight generations; deoxyadenosine (50 pg/ml) was added during labeling of reg84§_organisms. These compounds were present at the same con- centration during further growth for four doubling periods with PDD, wash by filtration or centrifugation, and inoculation and cultiva- tion in fresh medium. Samples were withdrawn from the resulting culture and analysed in triplicate by liquid scintillation counting as previously described (4). Alkaline Sucrose Sedimentation of DNA In experiments designed to define the nature of P00- induced damage to DNA, cells were grown with PDD in the presence of [3H] thymine (see above) and then washed by filtration. The fila- ments were then suspended in medium containing excess unlabeled thymine (50 ug/ml) and incubation was continued. Samples were removed at intervals, converted to spheroplasts, and then lysed on top of alkaline sucrose gradients by the method of Rupp and Howard-Flanders (31). In studies concerned with the nature of DNA synthesized during fragmentation, filaments were generated with unlabeled thymine, washed by centrifugation, and suspended in PDD-free medium containing [3H]thymine (25 uCi/ml). Samples were removed periodically and lysed by the method of Sedgwick (32). 65 Alkaline sucrose gradients (5.3 ml), containing 0.3 ml of a 50% alkaline sucrose shelf, were prepared (32) with an ultragrad grad- ient mixer (LKB Instrument Co., Bromma, Sweden). Gradients were centrifuged at 73,000 x g_for 120 min at 20°C in a Spinco SN 50.1 rotor (Beckman Instrument Co., Palo Alta, CA). Samples of 0.2 ml were collected from the top using a model 640 density gradient fractionator (ISCO, Lincoln, NB) with a flow rate of 0.5 ml per min. Coliphage T4 were layered on top of the gradients as a standard; the position of T4 in the gradient was measured by absorbance at 254 nm. 66 Table 1. Bacterial strains. Strain No. Genotype (3) Source w3110 thyA36 deoCZ . Bachmann p3478 as w3110, also polAl . Bachmann A82497 F'thr-l 1eu-6 thi-l argE3 . Bachmann his—4 proA2 thyAlZ deoBl6 lach galKZ mtl-l xyl-5 ara-l4 strA3l tsx-33 supE44 ABZSOO as A82497 except thyAlS, . Bachmann also uvrA6 A82487 as A82497, also recAl3 . Bachmann JC4583 F- Gal' Bl- Sm5 . Barbour JC4584 as JC4583, also . Barbour recBZl recC22 his- RESULTS Effect of P00 on Survival PDD did not significantly reduce viability of wild type cells for at least 4 h at the dose used to promote filamentation (5 pg/ml). In contrast, the time of incubation required to reduce the colony-forming ability of polA, recB,C, uvrA, and EggA_organisms to e'1 was 200, 120, 25, and 5 min, respectively (Figure l). PDD-Induced Filmentation After growth for 4 h with PDD, about 80% of wild type, 2915, and ugrA cells existed as filaments of at least 20 pm in length. About 50% of I§£§4£ cells formed similar filaments; this value was increased to 80% upon enrichment by filtration. Fila— mentation of rggA_mutants grown with PDD (5 ug/ml) was not detected. As shown by observation of washed cells in agar slide cultures, all bacteria that had not undergone filamentation during prior growth with PDD were dead as judged by their inability to increase further in mass or undergo cell division. Fragmentation of PDD-Induced Filaments The majority of washed filaments of wild type and 901A cells eventually underwent fragmentation in agar slide cultures. This process was delayed and often incomplete in slide cultures prepared from recB,C and uvrA filaments. Initial divisions 67 68 Figure l. The effect of Ej§yplatinum(II)diamminodichloride on the viability of cultures of wild type (0), [301A (A), recB,C (I), uvrA (0), and recA (O) E. coli K-12. 69 du-qu- - -——:-—fi- =-:-—— — =-:—-4d =—fi——_— — T 1 O. I 1' 1|” : F-pnhb - Eb-PF b I? ~:~-- _ P _ =—-Pb b P Ebb-hp - p 8 7 6 5 O O 0 O 4O _E\m:§ 3:52 .323 14 A 12 #0 3m time (h) 70 generally occurred in filaments in contact with or surrounded by other organisms; once initiated, the process of fragmentation con- tinued over a period of 3 to 5 h. Daughter cells arising after a filament had undergone three or four septation events thereafter underwent rapid division yielding bacteria of normal length. The patterns of fragmentation obtained in aerated liquid medium were distinct from those observed in agar slide cultures and were dependent upon the method used to remove PDD from filaments. After wash by filtration, the lag between innoculation in fresh medium and the onset of fragmentation was about 1, 2, and 4 h respectively for filaments of wild type, 9915, and EEEEEE_organisms. Filaments of gv§A_derivatives failed to divide during incubation for at least 24 h. In contrast, after wash by centrifugation and storage in cold buffer, the lag between inoculation and initial division was about 3, 4, 6, and 9 h respectively for filaments derived from wild type, polA, recB,C, and uvrA cells (Figure 2). Significant increases in the viability of filaments of DNA repair- deficient mutants occurred during these periods indicating the operation of some process of physiological rescue. This process, in fact, was essential for the onset of fragmentation in filaments of gy§A_organisms. Removal of PDD by centrifugation followed by storage in cold buffer resulted in fragmentation of at least 90% of filaments generated from wild type and DNA repair-deficient mutants. 71 Figure 2. Rescue of colony-forming ability by filaments of wild type (0), 901A (A), recB,C (I), and uvrA (0) E, coli K-12 after wash storage for 4 h at 4°C in 0.033 M potassium phosphate buffer, pH 7.0, and sus- pension in fresh medium. The dashed line illustrates the rate of growth for cells grown without gjs: platinum(II)diamminodichloride. 72 =::.+ l4 :5“... _ 44.3%.. 4:1... . 5:... I J l L J L Ii 1 J 5:... E ..:...H . E... E p.....hr. EZFO 9 8 7 6 5 mu. nlu nlu nlv mw ._E\m:c: 058.8 .828 time (h) 73 Net Synthesis of DNA Cells were grown with [3H]thymidine for eight to ten generations and then subcultured with the radioisotope in the presence and absence of PDD. Rates of net increase in trichloro- acetic acid-insoluble radioactivity were similar in both cases and paralleled increases in cell mass. After wash by filtration and dilution into PDD-free medium with [3H]thymidine, the DNA and cell mass in cultures derived from wild type, 9915, and [EEE,E_strains continued to increase without lag or change in ratio. Following this procedure, however, there was no net increases in DNA or cell mass of suspended filaments of uy§A_cells. In contrast, after wash by centrifugation and storage in cold buffer, a residual period of net DNA synthesis occurred in all DNA repair-deficient mutants followed by a significant lag (Figure 3). Net synthesis of DNA again commenced at a time corresponding to the onset of fragmen- tation. Characterization of PDD-Induced Chromosomal Alterations Wild type cells were grown with [3H]thymine for four doubling times with various concentrations of PDD. Upon centri- fugation in alkaline sucrose gradients, DNA released from fila- ments cultivated with 20 pg or more PDD/m1 exhibited a significant reduction of radioactivity in fractions of normal chromosomal molecular weight and progressive increase in fractions of low molecular weight (Figure 4). Identical results were obtained with polA, recB,C (not illustrated), and uvrA filaments (see below) grown 74 Figure 3. Net synthesis of deoxyribonucleic acid in filaments of wild type (a), recB,C (b), polA (c), and uvrA (d) _E. coli K-12 after wash and storage for 4 h at 4°C in 0.033 M potassium phosphate buffer, pH 7.0. 75 ”hr! 1 1 I JLIIIJI l L Tlllllj T T LLLIJJ L I 1'0 0 14%;L11 l l O 1 I TVVT—T I WITTTTI I I ITTj 1] r1 1 TTTI . d .0 JIIJLJ J I j IIITITT I 1 IO lllllll l_-IO 1 _._1J N (QOI) WHO I 9 time (h) Figure 4. 76 Sedimentation profiles in alkaline sucrose gradients of deoxyribonucleic acid (DNA) released from filaments of E, coli N3110 (wild type) after growth for four doubling times in the presence of 0 (a), 10 (b), 20 (c), 30 (d), 40 (e), and 50 (f) pg of gigfplatinum(11)diammino- dichloride per ml of medium. Arrows indicate the loca- tion of bacteriophage T DNA used a standard; zero cor- responds to the top of Ihe gradient. 77 IS 20 25 3O 5 IO 0 U 1 ‘2 Mgngsooogpm IDIOT )0 wowed 101‘ 4 ID fraction no. 78 with 5 pg of PDD per ml. These findings indicate that the accumu- lation of single strand breaks in DNA is a major consequence of exposure to P00. Not all such breaks necessarily arose as a func- tion of excision repair since DNA from Evgfl_filaments grown with 5 pg of PDD per ml contained both light and heavy fractions typical of those containing interstrand cross-links (9) (Figure 5). When .gyEA_cells were grown for only one doubling period with P00 (20 pg/ml) to minimize replication repair, the normal DNA sedi- mentation profiles were maintained whereas an increased light fraction was observed in the DNA profile of wild type organisms (Figure 6). Accordingly, some single strand breaks, especially those in DNA of ug:A_filaments after prolonged growth with PDD, may have occurred during replication over unexcised damaged bases. DNA of EggA_cells grown with 5 pg or more of PDD per ml underwent pronounced degradation to trichloroacetic acid-soluble fragments (not illustrated). Repair of PDD-Induced Chromosome Damage We examined the possibility that PDD-induced single strand breaks in DNA of filaments underwent repair during the lag between removal of the compound and the onset of fragmentation. As shown in Figure 7, a DNA sedimentation profile similar to that of control cells was recovered upon centrifugation in alkaline sucrose after filtration-washed filaments of wild type cells had undergone incu- bation for 60 min and fragmentation had begun. Similarly, restora- tion of normal profiles occurred in DNA of filtration-washed polA 79 Figure 5. Sedimentation profiles in alkaline sucrose gradients of deoxyribonucleic acid released from filaments of E. coli A82500 (uvrA) after growth for four doubling times in the presence of 0 (a), 5 (b), 10 (c), and 20 (d) pg of Ejg:platinum(II)diamminodichloride. Arrows indicate the position of the standard. percent of total radioactivity 80 1 4 1 L b I r 1 1 T’ TT 1- -1 F- ) - a 1 1 .1 1 w 0 5 10 I 20 25 3O fraction no. Figure 6. 81 Sedimentation profiles in alkaline sucrose gradients of deoxyribonucleic acid released from E. coli w3110 (wild type) (C) and A82500 (uvrA) (A) after growth for one doubling period with 5 pg/ml Ej§7platinum(II)- diamminodichloride. The arrows indicate the position of the standard. percent of total radioactivity 6'1 5 01 82 l 00", 1 I l I 5 IO 15 20 25 fraction no. 30 Figure 7. 83 Sedimentation profiles inalkaline sucrose gradients of deoxyribonucleic acid isolated from E, coli N3110 (wild type) grown without gj§7platinum(II)diamminodichloride (a) and profiles after growth for four doubling times with the compound (20 pg/ml), wash by filtration, and incubation in fresh medium after 0 (b), 30 (c), and 60 min (d). Arrows indicate the position of the T4 marker. percent of total radioactivity 84 I?! 1.1.: 10 IS 202530 fraction no. 85 and rggE,E_filaments at the time fragmentation commenced (not illustrated). In order to permit comparison of all DNA repair- deficient mutants, the sedimentation profiles of DNA synthesized after removal of PDD by centrifugation and storage at 4°C were determined. In all cases, a significant percentage of the DNA had achieved a sedimentation rate equivalent to that of the normal chromosome at the time fragmentation commenced (Figure 8). Repair of breaks was essentially complete when 90% of the fragments had undergone at least one division. At this time slowly sedimenting DNA was detected suggesting the presence of new division forks. Figure 8. 86 Sedimentation profiles in alkaline sucrose gradients of deoxyribonucleic acid released from wild type (a), polA (b), recB,C (c), and uvrA (d) isolates of E, coli K-12 after growth for four doubling times with Ejé: platinum(II)diamminodichloride, wash by centrifugation and storage at 4°C for 4 h, and inoculation into fresh medium containing 3H-thymine. Samples of each mutant were prepared 20 min after inoculation (O), at the onset of fragmentation (A), and at the time when the number of filaments was no more than 10% of the total population (I) (see Results). Arrows indicate the presence of internal standard. 87 mm ON Tm. .0: SEE: Om mm CW 0. I: L It) I Q 1 9 O N Kunuooogpoi mo; ;o wowed .1 Q J “2 1 O N DISCUSSION The concentration of PDD used to induce filamentous growth of E, coli K-12 (5 pg/ml) reduced the colony-forming ability of DNA repair-deficient mutants, especially that of recB,C, uvrA, and recA isolates. The latter, in fact, underwent reckless degradation of DNA resulting in death before filamentous growth could occur. PDD also promoted dose-dependent accumulation of single strand breaks in chromosomes of the other tested DNA repair-deficient and wild type organisms as judged by profiles recovered in alkaline sucrose gradients. After wash of filaments by either filtration or centri- fugation and storage at 4°C, only wild type or pglA_filaments underwent complete fragmentation in agar slide cultures. These isolates were also more resistant to the lethal effect of PDD than were E§E§;Q.°V gy§A_organisms. After wash by filtration and sus- pension in liquid medium, fragmentation of wild type and pglA_fila- ments occurred more promptly; this process also promoted cell divi- sion in filaments of 5gg§,§_but not 2355 mutants. The latter, however, underwent fragmentation after wash by centrifugation and storage at 4°C. Removal of PDD by this method delayed the onset of fragmentation by 2 h in wild type, 9915, and EEEE,E_isolates but‘ was essential for the occurrence of cell division in EgyA_filaments. Filaments of all DNA repair-deficient mutants were capable of recovering significant colony-forming ability during the lag 88 89 prior to the onset of fragmentation. Although the nature of this mechanism of physiological rescue was not defined, it appeared to be dependent upon storage of filaments in the cold and then sus- pension in aerated fresh medium at an optical density of at least 0.3. The latter requirement may reflect the observation that frag- mentation in agar slide cultures first occurred among filaments in close contact with neighboring organisms. Similar neighbor restor- ation effects have been described (1, 2, 37). Return of temperature- sensitive mutants blocked in cell division to permissive conditions can result in rapid fragmentation of filaments into cells of nearly normal size (6, 24, 26). This pattern was not detected upon removal of P00 where fragmentation proceeded erratically over a period of 3 to 5 h. The delay between removal of PDD and the initiation of cell division was dependent upon the ability of the bacteria to repair damaged DNA. Organisms with mutations that reduced ability to ligate gapped DNA (polA, recB,C) or assured the presence of gaps in newly synthesized DNA (gygfl) remained as filaments after wild type cells had initiated cell division. This result would be expected if fragmentation was dependent upon ligation of PDD-induced gaps, a step that is necessary for subsequent generation of super coiled nucleoid structure (38). Of interest in this context was the find- ing that nalidixic acid, which inhibits the activity of DNA gyrase (35), both decreased colony-forming ability and induced filamentous growth in recombination deficient mutants. The latter were also able to divide after removal of the compound (L. S. McDaniel, and 90 N. E. Hill, Abstr. Annu. Mtg. Amer. Soc. Microbiol. 1979, H25, p. 123). Similarly, temperature-sensitive mutants blocked in DNA ligation often exhibit reduced ligase activity under permissive conditions where a high percentage of the cells exist as filaments (10). These observations all favor the hypothesis that maintenance of supercoiled chromosome structure is requisite for cell division. The erratic onset of fragmentation in PDD-induced filaments observed in this study may reflect initiation of division at septation sites adjacent to newly repaired chromosomes. Subsequent restoration of normal structure in other nucleoids would result in occurrence of additional divisions. The observation that a period of storage at 4°C was neces- sary to permit fragmentation of gy[A_filaments was not expected. Storage at higher temperatures for the same period, with or without an energy source, did not mimic this effect. Use of cold storage with DNA repair-deficient but not wild type filaments resulted in delay of new DNA synthesis after inoculation into fresh medium. Possibly this lag before initiation of new rounds of replication was essential for the elimination of PDD-induced damage by recombi- nation repair or by excision via some reaction other than that catalyzed by the uvrA product (23). ACKNOWLEDGMENTS We thank Janet Fowler for technical assistance and Doris J. Beck for supplying gigfplatinum(1I)diamminodichloride. We greatly acknowledge help received from Richard M. Moore who initially observed the occurrence of sheared DNA in bacteria treated with PDD. This paper has been assigned Journal Article No. 9299 of the Michigan Agricultural Experiment Station. 91 LITERATURE CITED Adler, H. I., N. 0. Fisher, A. A. Hardigree, and G. E. Stapleton. 1966. Repair of radiation-induced damage to the cell division mechanism of Escherichia coli. J. Bacteriol. 91, 737-742. Allen, J. S., C. C. Filip, R. A. Gustafson. R. G. Allen, and J. R. Nalker. 1974. Regulation of bacterial cell divi- sion: genetic and phenotypic analysis of temperature sensitive, multinucleate, filament forming mutants of Escherichia coli. J. Bacteriol. 111: 978-986. Bachmann, B. J., K. Brooks Low, and A. L. Taylor. 1976. Recalibrated linkage map of Escherichia coli K-12. Bacterial. Rev. 49; 116-167. Beck, D. J., and R. R. Brubaker. 1973. Effect of Eiéf platinum(II)diamminodichloride on wild type and deoxy- ribunucleic acid repair deficient mutants of Escherichia coli. J. Bacteriol. 11g; 1247-1252. Beck, D. J., and R. R. Brubaker. 1975. Mutagenic properties of cis-platinum(II)diamminodichloride in Escherichia coli. Mutat. Res. El; l8l-189. Castellazzi, M., J. George, and G. Buttin. 1972. Prophage induction and cell division in E, coli. 1. Further characterization of the thermosensitive mutation tjj}l whose expression mimics the effect of u.v. irradiation. Mol. Gen. Genet. 112; 139-152. Castellazzi, M., J. George, and G. Buttin. 1972. Prophage induction and cell division in E, coli. II. Linked (recA, Egg) and unlinked (lgx) suppressors of tis-l mediated induction and filmentation. Mol. Gen. Genet. 119, 153-174. Cohen, G. L., N. R. Bauer, J. K. Barton, and S. J. Lippard. 1979. Binding of gj§_and trans-dichlorodiammine platinum II to DNA. Evidence for unwinding and shortening of the double helix. Science 29;; 1014-1016. 92 10. ll. 12. l3. 14. 15. 16. 17. l8. 19. 93 Cole, R. S. 1973. Repair of DNA containing interstrand cross- links in Escherichia coli: sequential excision and recombination. Proc. Natl. Acad. Sci. U.S.A. 19; 1064-1068. Gellert, M., and M. L. Bullock. 1970. DNA ligase mutants of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. E1: 1580-1587. George, J., M. Castellazzi, and G. Buttin. 1975. Prophage induction and cell division in E. coli. III. Mutations stjA and gjjB restore cell division in Ejf_andllgg strains and premit expression of mutator properties of Eli. Mol. Gen. Genet. 149; 309-332. Grossman, L., A. Braun, R. Feldberg, and I. Mahler. 1975. Enzymatic repair of DNA. Ann. Rev. Biochem. 44; l9-43. Harder, H. C., and B. Rosenberg. 1970. Inhibitory effects of antitumor platinum compounds on DNA, RNA, and protein synthesis in mammalian cells ifl_vitro. Int. J. Cancer E, 207-216. Howle, J. A., and G. R. Gale. 1970. gig-Dichlorodiammine- platinum(II): persistent and selective inhibition of deoxyribonucleic acid synthesis jg_vivo. Biochem. Pharmacol. 12; 2757-2762. Lecointe, P., J. P. Macquet, J. L. Butour, and C. Paoletti. 1977. Relative efficiencies of a series of square- planar platinum(II) compounds on Salmonella mutagenesis. Mutat. Res. 48; 139-144. Leopold, N. R., E. C. Miller, and J. A. Miller. 1979. Car- cinogenicity of antitumor gig-platinum(II) coordination complexes in mouse and rat. Cancer Res. 39; 913-918. Little, J. N., and D. G. Kleid. 1977. Escherichia coli protein X is the recA gene product. J. Biol. Chem. Egg: 6251-6252. Markham, B. E., and R. R. Brubaker. 1978. Fragmentation of gj§7platinum II diamminodichloride-induced filaments of wild type and deoxyribonucleic acid repair-deficient mutants of Escherichia coli. Biochemie E9; 1050-1052. Moore, R. L., and R. R. Brubaker. 1976. Effect of Eiér platinum(II)-diamminodichloride on cell division of Hyphomicrobium and Caulobacter. J. Bacteriol. lEE, 317-323. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 94 Neidhardt, F. C., P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. J. Bacteriol. TEE, 317-323. Pascoe, J. M., and J. J. Roberts. 1974. Interactions between mammalian cell DNA and inorganic platinum compounds. I. DNA interstrand cross-linking and cytotoxic properties of platinum(II) compounds. Biochem. Parmacol. g;: 1345- 1357. Radman, M. 1975. SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis, p. 355-367. ln_P. Hanawatt and R. B. Setlow (ed.), Molecular Mechanisms for Repair of DNA, part A. Plenum Press, New York. Radman, M. 1976. An endonuclease from Escherichia coli that introduces single polynucleotide chain scissions in ultraviolet irradiated DNA. J. Biol. Chem. EEl; 1438- 1445. Reeves, J. N., D. J. Graves, and D. J. Clark. 1970. Regula- tion of cell division in Escherichia coli: characteri- zation of temperature sensitive mutants. J. Bacteriol. 11g; 314-322. Reslova, S. 1971/72. The induction of lysogenic strains of Escherichia coli by Eig-dichlorodiammineplatinum(II). Chem. Biol. Interact. 4: 66-70. Richad, M., and Y. Hirota. 1973. Processes of cellular divi- sion in Escherichia coli: physiological study on thermosensitive mutants defective in cell division. J. Bacteriol. llg: 314-322. Roberts, J. J., and J. M. Pascoe. 1972. Cross—linking of complementary strands of DNA in mammalian cells by antitumor platinum compounds. Nature (London) EEE, 282-284. Robbins, A. B. 1973. The reaction of 14C-labelled platinum ethylenediammine dichloride with nucleic acid consti- tuents. Chem. Biol. Interact. E, 35-44. Rosenberg, B., E. Renshaw, L. Van Camp, J. Hartwick, and J. Brobnik. 1967. Platinum-induced filamentous growth in Escherichia coli. J. Bacteriol. 2;, 716-721. Rosenberg, B., and L. Van Camp. 1970. Successful regression of large solid Sarcoma 180 tumors by platinum compounds. Cancer Res. g9; 1799-1802. 31. 32. 33. 34. 35. 36. 37. 38. 95 Rupp, w. 0., and P. Howard-Flanders. 1968. Discontinuities in DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J. Mol. Biol. 31: 291-304. Sedgwick, S. G. 1975. Genetic and kinetic evidence for different types of postreplication repair in Escherichia coli B. J. Bacteriol. 1g}; 154-161. Shimuzu, M., and B. Rosenberg. 1973. A similar action to UV-irradiation and preferential inhibition of DNA synthesis in E, coli by antitumor platinum compounds. J. Antibiotics 26: 243-245. Shooter, K. V., R. Howse, R. K. Merrifield, and A. 8. Robbins. 1972. The interaction of platinum(II) compounds with bacteriophage T7 and R17. Chem. Biol. Interact. E: 289-307. Sugino, A., C. L. Peebles, K. N. Kreuzer, and N. Cozzarelli. 1977. Mechanism of action of naldixic acid: purifica- tion of Escherichia coli nalA gene product and its rela- tionship to DNA gyrase and a novel nick-closing enzyme. Proc. Natl. Acad. Sci. U.S.A. 24, 4767-4771. Taylor, R. T., J. H. Carver, M. L. Hanna, and D. L. Wanders. 1979. Platinum-induced mutations to 8-azaguanine resistance in chinese hamster ovary cells. Mutat. Res. E2; 65-80. Witkin, E. M. 1976. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacterial. Rev. 49; 869-907. Worcel, A., and E. Burgi. 1972. On the structure of the folded chromosome of Escherichia coli. J. Mol. Biol. .21: 127-147. SECTION IV 96 Effect of the Protease Inhibitor Antipain on Recovery of Division Potential in Eig-Platinum(II)diamminodichloride-Induced Filaments of Escherichia coli K-12 B. E. Markham* and R. R. Brubaker Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 *Present address: Department of Microbiology College of Medicine University of Arizona Tuscan, Arizona 85724 97 ABSTRACT Cultivation of Escherichia coli with gjg-platinum(II)diammino- dichloride (PDD) is known to promote filamentous growth associated with maintenance of gapped DNA. Otherwise nontoxic concentrations of the protease inhibitor antipain, supplied with PDD, decreased viabil- ity, prevented filamentation, and reduced the ability of cells to remove interstrand crosslinks. Nhen added after removal of PDD, anti- pain prolonged the onset of fragmentation of filamented cells. 98 99 Exponentially grown cells of Escherichia coli N3110 (thyA36 gggCZ) were inoculated and cultivated as previously des- cribed (8) in supplemented defined medium (12) containing increasing concentrations (0 to 5.0 mM) of the protease inhibitor antipain [(l-carboxy-Z-phenylethyl)carbamyl-L-arginyl-L-valylargininal] (16). At the levels of 0.2 mM or more, the inhibitor significantly reduced doubling times and net generation of cell mass (Figure la). In the presence of gig-platinum(II)diamminodichloride (PDD) (5 pg/ml), an agent known to promote expression of SOS functions including fila- mentous growth (2, l3, l4), otherwise nontoxic levels of antipain (Figure lb) severely reduced rates of increase in optical density and maximum absorbancy. This synergistic effect was anticipated because antipain had been reported to inhibit thermally-induced filamentation (10, ll). Antipain at concentrations of 0.1 mM or less did not influence colony-forming ability although at levels of 0.2 mM (Figure 2) or more (not illustrated) the inhibitor became pro- gressively more toxic. Cells grown for 4 h with 0.1 mM antipain were of normal size whereas at concentrations of 0.2 mM the organ- isms were about twice their normal length. Cells grown with P00 (5 pg/ml) alone for 4 h existed primarily as multinucleated fila- ments of at least 30 pm in length; colony-forming ability was reduced by about 20% during this period. In contrast, the environ- ment containing marginally lethal concentrations of both PDD (5 pg/ml) and antipain (0.1 or 0.2 mM) was extremely toxic result- ing in exponential decreases in colony-forming ability after Figure l. 100 Increase in optical density at 620 nm of E, coli N3110 incubated at 37°C in defined medium containing no addi- tion (0), both frames and (a) 0.05 mM (A), 0.1 mM (I), 0.2 mM (0), 0.5 mM (0) and 5 mM (0) antipain and (b) PDD (5 pg/ml) alone ((3) and PDD with 0.1 mM (2’).) or 0.2 mM (0) antipain. 101 .6: Cum 33:3 60:8 --- - q - fi-—-1—— - flu .l T 1 r- I. r I. I: l b hPPPLP P P —PPhr—b F qqd-— — - d--_1— ' Q I l o J .. .4 r / I c r...w. . . . .p...rp _ m time (h) Figure 2. 102 Colony-forming ability of E, coli N3110 incubated at 37°C in defined medium containing no additions (O), 0.1 mM antipain (A), 0.2 mM antipain (I), PDD (5 pg/ml) ((3), P00 (5 pg/ml) plus 0.1 mM antipain (o), and PDD (5 pg/ml) plus 0.2 mM antipain (0). 103 L O. 6 ._E\m:§ 056.8 .323 o. no. time (h) 104 incubation for 1 h [a period of time probably required for signifi- cant uptake of P00 (1)]. Microscopic examination of these cultures after 4 h of incubation revealed that the number of total cells had approximately doubled and that filamentous growth had not occurred. To determine if combined use of PDD and antipain promoted reckless degradation of deoxyribonucleic acid (DNA) in wild type cells, as occurs with [EEA cells treated with PDD alone (1), cells were labeled with [3H]-thymine (20 pCi/ml) and then the percentage of remaining trichloroacetic acid-insoluble counts was determined upon transfer to medium containing excess unlabeled thymine (5). No significant loss occurred after transfer to medium containing antipain (0.1 or 0.2 mM) alone; a maximum loss of about 25% was detected in cultures containing PDD (5 pg/ml) alone or in those containing both compounds. These results indicate that cell death promoted by PDD plus antipain did not reflect unrestricted degrada- tion of DNA. The possibility remained, however, that combined treatment with PDD and antipain might result in other types of chromosomal damage. To test this possibility, cells previously labeled by growth for 4 generations with [3H]-thymine (20 pCi/ml) were culti- vated for 4 doubling times with excess unlabeled thymine and P00 (5 pg/ml), antipain (0.1 mM), or both. The organisms were then lysed on top of alkaline sucrose gradients and centrifuged as pre- viously described (8). Profiles of DNA recovered from untreated control cells and those grown with antipain alone were similar (Figure 3a and 3c). The profile obtained from PDD-induced Figure 3. 105 Profiles an alkaline sucrose gradients on DNA from E. coli N3110 grown for 4 doubling periods with T3Hl-thymine and no other addition (a), PDD (5 pg/ml) (b), 0.1 mM antipain (c), and PDD (5 pg/ml) plus 0.1 mM antipain (d). Arrows indicate the presence of T4 bacteriophage used as an internal marker, zero corresponds to the top of the gradient. 106 . _ . d . i O a 3 5 .. i i 2 0 i .l .I 2 1 .. .. :13 1 i i w .11 1 1 5 C d p b n n p p p m 5 m o 5 m m 3.280060. .22 .o 23.8 fraction no. 107 filaments (Figure 3b) indicated the presence of multiple single- strand breaks as previously reported (8). In contrast, the DNA obtained from cells grown with both antipain and PDD (Figure 3d) had significantly less single strand breakage and possessed a shoulder of fast-sedimenting fractions indicative of interstrand crosslinked DNA (3, 15) similar to that observed in excision- deficient (gyrA) E, gglj_incubated with PDD alone (8). Cells were grown for 4 doubling periods with PDD (5 pg/ml) and the resulting filaments were washed by filtration and suspended in fresh medium containing 0.1 mM or no antipain. As judged by direct microscopic observation and determination of colony-forming units per ml, filaments in the control culture initiated cell divi- sion after 1 h of incubation (Figure 4). In contrast, filaments incubated with antipain required about 4 h of incubation before undergoing fragmentation. To determine if this antipain-dependent lag in cell division was associated with delay in repair of PDD-induced damage to DNA, filaments were prepared by growth for 4 doubling periods in [3H]- thymine (20 pCi/ml). After wash by filtration (8), the filaments were incubated with or without 0.1 mM antipain in excess unlabeled thymine; samples were removed and prepared for centrifugation an alkaline sucrose gradients at intervals of 30 min. As reported previously (8), filaments from the culture lacking antipain com- pleted repair within the first hour of incubation after removal of P00 (not illustrated). The repair of PDD-induced damage to DNA was also completed within 1 h in antipain-treated filaments as Figure 4. 108 Colony-forming ability of PDD—induced filaments, washed by filtration, during incubation at 37°C in fresh medium in the absence (0) or presence of 0.1 mM antipain (A). 109 :____ _ ::_:__ :_:.d_ _ fi 10 fil I. .l I; j .1 1| .1 1 J l J rl I. F...- _ _ .ZEF .P p F ::Pb_ — _ b m 9 8 7 m m nlu m _E\m:c: 9.32.0. .828 time (h) 110 shown by identical decrease in slow sedimenting fractions with concomitant increase in counts co-sedimenting with control DNA (Figure 5). These findings provide some insights into the nature of mechanisms involved in control of cell division and regulation of SOS functions. Previously reported findings (8) indicated that completion of repair of PDD-induced damage to DNA was necessary to initiate cell division. The results presented in this report suggest that cell division may also depend upon expression of proteolytic activity because fragmentation but not repair of DNA was significantly delayed by antipain in washed PDD-treated fila- ments. An obvious candidate for inhibition by antipain is the Eggfl_ product, protein X, which is induced during filamentous growth and is capable of autoregulation (4, 7, 9). Other possibilities, how- ever, may exist. For example, filamentous lgg_mutants of E, £911 are Deg" and thus unable to degrade certain protein fragments (6, 17). This capability, which is evidently essential for the onset of cell division in filaments, might be inhibited by antipain. Further study into the role of antipain in maintaining lethal PDD- induced interstrand crosslinks in Eygf E, ggli_may resolve this problem. 111 Figure 5. Profiles an alkaline sucrose gradients of DNA from filtration-washed filaments of E, coli grown for 4 doubling periods with [3H]-thymine and P00 (5 pg/ml) during incubation at 37°C for 0 (a), 30 (b), and 60 min (c) in fresh medium containing 0.1 mM antipain. Arrows indicate the presence of T4 bacteriophage used as an internal marker; zero corresponds to the top of the marker. 112 B 20 25 3O 15 IO 5 15-0 IO- Is-b 23.80060. .22 .0 2.8.3 fraction no. LITERATURE CITED Beck, D. J., and R. R. Brubaker. 1973. Effect of gis- platinumII-diamminodichloride on wild type and deoxy- ribonucleic acid repair-deficient mutants of Escherichia coli. J. Bacteriol. 1§§, 1167-1170. Beck, 0. J., and R. R. Brubaker. 1975. Mutagenic properties of gig-platinum(II)diamminodichloride in Escherichia coli. Mutat. Res. El: 181-189. Cole, R. S. 1973. Repair of DNA containing interstrand cross- links in Escherichia coli: sequential excision and recombination. Proc. Natl. Acad. Sci. U.S.A. 20: 1064-1068. Emmerson, P. T., and S. C. West. 1977. Identification of protein X of Escherichia coli as the EEEAT/Eiff gene product. Mol. Gen. Genet. lEE: 77-85. Ferber, D. M., and R. R. Brubaker. 1979. Made of action of pesticin: N-acetylglucosaminidase activity. J. Bacteriol. lgg; 495-501. Gottesman, S., and D. Zipser. 1978. Deg phenotype of Escherichia coli lon mutants. J. Bacteriol. 1§§: 844-851. Gudas, L. J., and 0. w. Mount. 1977. Identification of the E925 (31f) gene product of Escherichia coli Proc. Natl. Acad. Sci. U.S.A. 23: 5280-5284. Markham, B. E., and R. R. Brubaker. 1980. Influence of chromosome integrity on cell division in gig-platinum(II)- diamminodichloride-induced filaments of wild type and deoxyribonucleic acid repair-deficient mutants of Escherichia coli. J. Bacteriol. (Submitted) McEntee, K. 1977. Protein X is the product of the E220 gene of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 24, 5275-5279. 113 10. 11. 12. 13. 14. 15. 16. 17. 114 Meyn, M. S., T. Rossman, P. Gottlieb, and w. Troll. 1978. Role of proteases in SOS regulation, p. 379-382. In P. C. Hanawalt, E. C. Freidberg, and C. F. Fox (edT), DNA Repair Mechanisms. Academic Press Inc., New York. Meyn, M. S., T. Rossman, and N. Troll. 1977. A protease inhibitor blocks SOS functions in Escherichia coli: antipain prevents A repressor inactivation, ultraviolet mutagenesis, and filamentous growth. Proc. Natl. Acad. Sci. U.S.A. 14, 1152-1156. Neidhardt, F. C., P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. J. Bacteriol. 112; 736-747. Reslova, S. 1971/72. The induction of lysogenic strains of Escherichia coli by Eig-dichlorodiammine platinum II. Chem.-Biol. Interact. 4; 66-70. Rosenberg, B., E. Renshaw, L. Van Camp, J. Harwick, and J. Drobnik. 1967. Platinum-induced filamentous growth in Escherichia coli. J. Bacteriol. 9;: 716-721. Van den Berg, H. N., and J. J. Roberts. 1975. Alkaline sucrose gradient studies: changes in the sedimentation profile of template DNA from chinese hamster V9-379A cells treated with the antitumor agent gig-platinum(II)- dianminodichloride. StudiaBiophys. E0: 39-46. Ningender, N. 1974. Proteinase inhibitors of microbial origin. A review, p. 548-559. In H. Fritz, H. Tschesche, L. J. Greene, and E. Fruscheit (Ed.), Proteinase Inihi- bition. Springer-Verlag, Berlin. Nitkin, E. M. 1976. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacterial. Rev. 49: 869-907. ACKNOWLEDGMENTS We thank Janet Fowler for technical assistance, Doris J. Beck for supplying PDD, and Paul A. Swenson for providing antipain. This paper has been assigned Journal Article No. 9300 of the Michigan Agricultural Experiment Station. 115