Strains of Escherichia coli possessing the pebAl mutation, a functional DNA polymerase I, and a temperature-sensitive mutation in DNA polymerase m can survive at the restrictive temperature (430C) for DNA polymerase m. The mutation rate of the bacterial genome of such strains after exposure to either UV light or ethyl methanesulfonate was measured by its rifampicin resistance or amino acid requirements. In addition, Weigle mutagenesis of preirradiated X phage was also measured. In all cases, no increase in mutagenesis was noted at the restrictive temperature for DNA polymerase HI. Introduction of a cloned DNA polymerase HI gene returned the mutation rate of the bacterial genome as well as the Weigle mutagenesis to normal at 43C. Using a recA-LacZ fusion, the SOS response after UV irradiation was measured and found to be normal at the restrictive and permissive temperature for DNA polymerase III, as was induction of X prophage. Recombination was also normal at either temperature. Our studies demonstrate that a functional DNA polymerase III is strictly required for mutagenesis at a step other than SOS induction.The functional DNA polymerase III holoenzyme complex of Escherichia coli is composed of at least seven proteins (1, 2). These components interact to replicate the E. coli genome at high fidelity. The a subunit of this complex possesses the polymerizing activity and is encoded by the dnaE (polC) gene (3). Temperature-sensitive mutations at this locus prevent replication at the restrictive temperature of 43°C (4). Mutations at the dnaE locus may produce strains with an increase in the spontaneous mutation rate (5) as well as UV-induced mutagenesis (6).The pcbAl mutation allows DNA polymerase I-dependent replication of the bacterial genome without a functional DNA polymerase III a subunit (7). Strains containing the pcbAl mutation, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III are viable at the restrictive temperature (43°C) for DNA polymerase III and, therefore, can be used to assess the role of DNA polymerase III in mutagenesis. Using such strains, we find that DNA polymerase III is required for mutagenesis of the bacterial genome by either UV irradiation or ethyl methanesulfonate (EtMes) exposure as well as for Weigle mutagenesis. DNA polymerase III is not required for induction of the SOS response or DNA recombination. MATERIALS AND METHODSStrains. The bacterial strains used are listed in Table 1. CSM61 and CSM14 are spontaneous, temperature-resistant revertants from HS432 (polAl, polB100, polC1O26, pcbAl) as previously described (7). CSM98 is a CSM61 derivative made polC+ by transduction. Strain ERli is an E486 (polC486) (8) derivative made by P1 transduction of a TnJO linked to pcbAl from RM552. Strain RM552 is an ESli (8) derivative containing TnJO linked to pcbAl (zic-J: :TnJO) transduced from a CSM61 derivative (7). Strain SB229 was constructed by transduction of recA56 srlJ300::TnJO into JM103.Plasmid pDS4-26 was provided by C. McHenry. It contains the coding r...

We have previously shown that some Escherichia coli [derivatives of strain HS432 (polAl, polB100, polC1026)] can replicate DNA at a restrictive temperature in the presence of a polCts mutation and that such revertants contain apparent DNA polymerase I activity. We demonstrate here that this strain of E. coli becomes temperature-resistant upon the introduction ofa normal gene for DNA polymerase I or suppression of the polAl nonsense mutation. Such temperature-resistant phenocopies become temperature-sensitive upon introduction of a temperature-sensitive DNA polymerase I gene. Our results confirm that DNA replication is DNA polymerase I-dependent in the temperature-resistant revertants, indicating that an alternative pathway of replication exists in E. coli. HS432 contains a transducible locus (which we term pebA) that can support an alternate pathway in other E. coli strains, so the effect of suppression ofpolCts is a general one.On the basis of available mutants and in vitro DNA replication systems, DNA replication in Escherichia coli appears to be a multistep process requiring a number of proteins (1, 2). Of We report here that DNA replication in the parent strain becomes temperature-resistant with introduction ofDNA polymerase I activity but is ts in the absence of DNA polymerase I or presence of a ts DNA polymerase I activity. We conclude that this strain contains a mutation (pcbA-) that allows replication to be dependent on DNA polymerase I polymerizing activity. This locus can be transduced to other E. coli strains and again exerts phenotypic suppression of the polCts mutation in the presence of DNA polymerase I. Our results indicate that E. coli has alternative pathways of DNA replication. MATERIALS AND METHODSBacterial Strains and Phage. E. coli strain HS432 (polA-, polB-, polC1026, his-, leu-) and its temperature-resistant derivative CSM61 (poLA-, polB-, polCts), and strains RK4604 (his-, lacam, nmtB-, metD-), E511 (polCts), and MJ245 (ValR, metE-) have been used previously (5). FTP439 (metE+, Vals, MeMeSR) was from E. Murgola (M. D. Anderson Hospital and Tumor Institute). Strains HS405 (polA12) and 108 (polA12) were from H. Shizuya (University of Southern California).Strain E486 (polA', polC486) was from C. McHenry (University ofTexas Health Science Center). Phage A i21nin5psu+2(s), q480 wild-type, and 480 psu+3 sus2 were from H. Ozeki (Kyoto University). Phage ON1 is a hybrid phage constructed by a cross between the two suppressor-carrying phages above, selected for h+w, i2l; it carries psu+3 and attes.Materials. Growth media were purchased from Difco. 3H-Labeled nucleoside triphosphates were purchased from Amersham/Searle. N-Ethylmaleimide (MalNEt) and methyl methanesulfonate (MeMeS) were purchased from Eastman.Culture Methods. Cells were grown in L broth. Selection for auxotrophic markers was done on M-9 medium. MeMeS resistance was measured as described (5). Temperature-resistant phenotype was measured by growth of duplicate L-broth plates at 320C and 420C. Val resistance was measured by ...

Escherichia coli possesses three DNA polyrnerases (17). DNA polymerase I has been implicated in DNA repair, since mutations at the polA locus increase sensitivity to many DNA-damaging agents, including UV irradiation (7), methyl methanesulfonate (MMS) (10), hydrogen peroxide (1), and bleomycin (25). No role has yet been discovered for DNA polymerase II. DNA polymerase III has a central role in DNA replication. Temperature-sensitive (ts) mutations of the dnaE (polQ gene coding for the a-subunit of DNA polymerase III are lethal at 43°C, the restrictive temperature (11,30). Cells lacking the a-subunit function of the holoenzyme are inviable, regardless of whether DNA polymerase I or II is present. Studies on the role of DNA polymerase III in DNA repair have been limited due to the inviability of the mutants at the restrictive temperature, but have implicated DNA polymerase III in excision and post-replication repair of UV damage (2,9,16,25,26,29), in repair of damage due to X rays (3,14,28), and possibly in recombination and mutagenesis (2-4, 13).The pcbAl genotype has been shown to suppress phenotypically three different polC(Ts) mutations if a functional DNA polymerase I is present (5,22,23 show an increase in sensitivity at 430C, where DNA polymerase III is inactive (5). This is compatible with our earlier observations suggesting a role in DNA repair for DNA polymerase III (9) and has led us to the current studies. In this paper, we report the results of testing these mutants for sensitivity to several different DNA-damaging agents. By quantitative cell survival as well as alkaline sucrose sedimentation, we show that DNA polymerase III is necessary for the optimal repair of MMS-and hydrogen peroxide-* Corresponding author.induced DNA damage but is not strictly required for the repair of damage by UV light, bleomycin, or psoralen. MATERIALS AND METHODS

Eschenichia coli cells with a polCb mutation contain a temperature-sensitive DNA polymerase III and fail to replicate DNA at the restrictive temperature (430C). Mutants deficient in polymerizing activity of the other two recognized DNA polymerases in E. coli can replicate DNA. We have isolated temperature-resistant revertants from a strain containing poIA-, polB , and poICt. mutations. These revertants grow at 430C, but analysis of partially purified DNA polymerase III from several such revertants shows a temperature-sensitive DNA polymerase III activity. Genetic analysis by P1 transduction confirms that such revertants can contain a JC, mutation and also a poLA-mutation. We find that such revertants behave phenotypically as PolI+ cells (DNA polymerase I-containing),and extracts of such cells show a DNA polymerase I-like activity.Revertants of poL4 , dnaAts and polA-, dnaBt, strains do not show such a DNA polymerase activity. The discovery and detailed analysis of a predominant DNA polymerase activity (DNA polymerase I) in Escherichia coil appeared to satisfy the requirement for an enzyme activity for replication (1). The isolation of a mutant defective in the polymerizing activity of this enzyme emphasized questions regarding its role. Subsequent studies have shown that it is required for optimal DNA repair activity after exposure to UV or x-ray radiation. DNA (3) identified that enzyme as required for replication and led to its model role as a replicase. The peculiar properties of DNA polymerase III which permit it to play a required role in replication, while the other DNA polymerases cannot, are not defined.We have isolated temperature-reistant mutants from a strain of E. colh containing polA -, polB-, and poiCts mutations.Analyzing such revertants, we were struck that a high percentage contain MalNEt-resistant DNA polymerase activity and exhibit PolI+ phenotype. This observation suggested to us either an unexpected pressure to select for polA + in poiCt,The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 Kadner (University of Virginia). Strain E511 (polCtb) has been used previously (3). HMS83 (polA , polBi) was from C. C. Richardson (Harvard Medical School). MJ245 (polA +, metE-) was from M. Murgola (M. D. Anderson Hospital and Tumor Institute).Isolation of Temperature-Resistant Revertants. Revertants of HS432 were selected by plating approximately 3 X 107 cells per plate on L agar from L-broth cultures grown at 320C. Plates were incubated at 430C for 2 days. Individual colonies were picked from among approximately 20-80 colonies per plate. These colonies were streaked again and checked for the temperature-resistant phenotype. On some occasions, a liquid culture started from an individual colony was plated and incubated at 430C and only one colony was selected per plate to ensure independent revertants. P1 Transduction. P1 transduction was done as described by Lennox (6). Strain R...

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