Subunit Of DNA Polymerase Epsilon Research Articles

EARLY IN SHORT DAYS 7(ESD7) encodes the catalytic subunit of DNA polymerase epsilon and is required for flowering repression through a mechanism involving epigenetic gene silencing

We have characterized a mutation affecting the Arabidopsis EARLY IN SHORT DAYS 7 (ESD7) gene encoding the catalytic subunit of DNA polymerase epsilon (epsilon), AtPOL2a. The esd7-1 mutation causes early flowering independently of photoperiod, shortened inflorescence internodes and altered leaf and root development. esd7-1 is a hypomorphic allele whereas knockout alleles displayed an embryo-lethal phenotype. The esd7 early flowering phenotype requires functional FT and SOC1 proteins and might also be related to the misregulation of AG and AG-like gene expression found in esd7. Genes involved in the modulation of chromatin structural dynamics, such as LHP1/TFL2 and EBS, which negatively regulate FT expression, were found to interact genetically with ESD7. In fact a molecular interaction between the carboxy terminus of ESD7 and TFL2 was demonstrated in vitro. Besides, fas2 mutations suppressed the esd7 early flowering phenotype and ICU2 was found to interact with ESD7. Discrete regions of the chromatin of FT and AG loci were enriched in activating epigenetic marks in the esd7-1 mutant. We concluded that ESD7 might be participating in processes involved in chromatin-mediated cellular memory.

Generation of Bradyrhizobium japonicum Mutants with Increased N 2 O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N(2)O) reductase (N(2)OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N(2)O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction into B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N(2)O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N(2)OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N(2)OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.

Mrc1 and DNA Polymerase ɛ Function Together in Linking DNA Replication and the S Phase Checkpoint

Yeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replication forks and a mediator of the S phase checkpoint. We report that Mrc1 interacts with Pol2, the catalytic subunit of DNA polymerase epsilon, essential for leading-strand DNA replication and for the checkpoint. In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halves of Pol2 (Pol2N and Pol2C). Strikingly, phosphorylation of Mrc1 during the S phase checkpoint abolishes Pol2N binding, but not Pol2C interaction. Mrc1 is required to stabilize Pol2 at replication forks stalled in HU. The bimodal Mrc1/Pol2 interaction may be an additional step in regulating the S phase checkpoint response to DNA damage on the leading strand. We propose that Mrc1, which also interacts with the MCMs, may modulate coupling of polymerization and unwinding at the replication fork.

Efficient silencing of the gene coding for the ɛ subunit of DNA polymerase III inEscherichia coliis triggered by antisense RNAs featuring stabilityin vivo

The Escherichia coli gene dnaQ coding for the epsilon subunit of DNA polymerase III was suppressed in vivo via antisense RNAs. To this aim, different fragments of dnaQ were cloned in reverse orientation into the pBAD-HisB vector or into pT3T7, and the corresponding antisense RNAs were conditionally expressed in vivo. Antisense transcripts featuring fast hybridization in vitro with dnaQ mRNA but lacking stability in vivo did not suppress the target gene. Moreover, the in vivo concentration of an antisense transcript was positively correlated to its silencing effectiveness.

Cell Cycle-dependent Phosphorylation of the DNA Polymerase Epsilon Subunit, Dpb2, by the Cdc28 Cyclin-dependent Protein Kinase

DNA polymerase epsilon (Polepsilon), one of the three major eukaryotic replicative polymerases, is comprised of the essential catalytic subunit, called Pol2 in budding yeast, and three accessory subunits, only one of which, Dpb2, is essential. Polepsilon is recruited to replication origins during late G(1) phase prior to activation of replication. In this work we show that the budding yeast Dpb2 is phosphorylated in a cell cycle-dependent manner during late G(1) phase. Phosphorylation results in the appearance of a lower mobility species. The appearance of that species in vivo is dependent upon the Cdc28 cyclin-dependent protein kinase (CDK), which can directly phosphorylate Dpb2 in vitro. Either G(1) cyclin (Cln) or B-type cyclin (Clb)-associated CDK is sufficient for phosphorylation. Mapping of phosphorylation sites by mass spectrometry using a novel gel-based proteolysis protocol shows that, of the three consensus CDK phosphorylation sites, at least two, Ser-144 and Ser-616, are phosphorylated in vivo. The Cdc28 CDK phosphorylates only Ser-144 in vitro. Using site-directed mutagenesis, we show that Ser-144 is sufficient for the formation of the lower mobility form of Dpb2 in vivo. In contrast, Ser-616 appears not to be phosphorylated by Cdc28. Finally, inactivation of all three CDK consensus sites in Dpb2 results in a synthetic phenotype with the pol2-11 mutation, leading to decreased spore viability, slow growth, and increased thermosensitivity. We suggest that phosphorylation of Dpb2 during late G(1) phase at CDK consensus sites facilitates the interaction with Pol2 or the activity of Polepsilon

Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach

The in vivo induced antigen technology (IVIAT) 1 has been used for the identification of open reading frames (ORFs) which could be possible therapeutic targets. A recombinant λgt11:: Mycobacterium tuberculosis H37Rv expression library was screened with pooled TB patient sera preabsorbed with in vitro grown M. tuberculosis H37Rv. Preabsorption of pooled TB patient sera allowed identification of antigens specifically expressed or upregulated during infection and growth in vivo. Six ORFs were identified, of which four ( rv0287, rv2402, rv3878 and rv1045) were of hypothetical functions. Rv0287 is a probable regulatory protein. Rv3878 is present uniquely in M. tuberculosis H37Rv and is a part of RDI deletion region of M. bovis BCG, which includes esat 6 region. This could be exploited as a tool for diagnosis. Two ORFs were assigned function solely on the basis of homology, dnaQ ( rv3711c) and lpdA ( rv3303c). dnaQ codes for the epsilon subunit of DNA polymerase III, which is responsible for the proofreading activity of the complex. lpdA codes for dihydrolipoamide dehydrogenase, which is a part of many multienzyme complexes such as pyruvate dehydrogenase, keto-acid dehydrogenase and α-ketoglutarate dehydrogenase. These two enzymes appear to be potential targets for drug development.

The DNA Polymerase Domain of polε Is Required for Rapid, Efficient, and Highly Accurate Chromosomal DNA Replication, Telomere Length Maintenance, and Normal Cell Senescence inSaccharomyces cerevisiae

Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase epsilon. This study investigates the cellular functions performed by the polymerase domain of Pol2p and its role in DNA metabolism. The pol2-16 mutation has a deletion in the catalytic domain of DNA polymerase epsilon that eliminates its polymerase and exonuclease activities. It is a viable mutant, which displays temperature sensitivity for growth and a defect in elongation step of chromosomal DNA replication even at permissive temperatures. This mutation is synthetic lethal in combination with temperature-sensitive mutants or the 3'- to 5'-exonuclease-deficient mutant of DNA polymerase delta in a haploid cell. These results suggest that the catalytic activity of DNA polymerase epsilon participates in the same pathway as DNA polymerase delta, and this is consistent with the observation that DNA polymerases delta and epsilon colocalize in some punctate foci on yeast chromatids during S phase. The pol2-16 mutant senesces more rapidly than wild type strain and also has shorter telomeres. These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast.

The second largest subunit of mouse DNA polymerase epsilon, DPE2, interacts with SAP18 and recruits the Sin3 co-repressor protein to DNA.

DNA polymerase epsilon is essential for cell viability and chromosomal DNA replication in budding yeast. In addition, DNA polymerase epsilon may be involved in DNA repair and cell-cycle checkpoint control. The enzyme consists of at least four subunits in mammalian cells as well as in yeast. The largest subunit of DNA polymerase epsilon is responsible for polymerase activity. To date, the functions of the other subunits have remained unknown. With a view to elucidating the functions of the second largest subunit of mouse DNA polymerase epsilon (DPE2), yeast two-hybrid screening was performed to identify mouse proteins that interact with this subunit. SAP18, a polypeptide associated with co-repressor protein Sin3, was identified as an interacting protein. A part of the N-terminal region of DPE2 (comprising amino acids 85-250) was found to be responsible for the interaction with SAP18. The interaction induced repression of transcription in reporter plasmid assays, which was inhibited by trichostatin A. These results indicate that DPE2 may recruit histone deacetylase (HDAC) to the replication fork to modify the chromatin structure.

Schizosaccharomyces pombe Cells Lacking the Amino-Terminal Catalytic Domains of DNA Polymerase Epsilon Are Viable but Require the DNA Damage Checkpoint Control

In Schizosaccharomyces pombe, the catalytic subunit of DNA polymerase epsilon (Pol epsilon) is encoded by cdc20(+) and is essential for chromosomal DNA replication. Here we demonstrate that the N-terminal half of Pol epsilon that includes the highly conserved polymerase and exonuclease domains is dispensable for cell viability, similar to observations made with regard to Saccharomyces cerevisiae. However, unlike budding yeast, we find that fission yeast cells lacking the N terminus of Pol epsilon (cdc20(DeltaN-term)) are hypersensitive to DNA-damaging agents and have a cell cycle delay. Moreover, the viability of cdc20(DeltaN-term) cells is dependent on expression of rad3(+), hus1(+), and chk1(+), three genes essential for the DNA damage checkpoint control. These data suggest that in the absence of the N terminus of Pol epsilon, cells accumulate DNA damage that must be repaired prior to mitosis. Our observation that S phase occurs more slowly for cdc20(DeltaN-term) cells suggests that DNA damage might result from defects in DNA synthesis. We hypothesize that the C-terminal half of Pol epsilon is required for assembly of the replicative complex at the onset of S phase. This unique and essential function of the C terminus is preserved in the absence of the N-terminal catalytic domains, suggesting that the C terminus can interact with and recruit other DNA polymerases to the site of initiation.

MDM2 interacts with the C-terminus of the catalytic subunit of DNA polymerase epsilon.

MDM2 is induced by p53 in response to cellular insults such as DNA damage and can have effects upon the cell cycle that are independent or downstream of p53. We used a yeast two-hybrid screen to identify proteins that bind to MDM2 and which therefore might be involved in these effects. We found that MDM2 can bind to the C-terminus of the catalytic subunit of DNA polymerase epsilon (DNA pol epsilon), to a region that is known to be essential in yeast. In an in vitro system we confirmed that MDM2 could bind to the homologous regions of both mouse and human DNA pol epsilon and to full-length human DNA pol epsilon. DNA pol epsilon co-immunoprecipitated with MDM2 from transfected H1299 cells and also from a HeLa cell nuclear extract. We show here that the DNA pol epsilon-interacting domain of MDM2 is located between amino acids 50 and 166. Our studies provide evidence that MDM2 interacts with a region of DNA pol epsilon that plays a critical role in the function of DNA pol epsilon.

Disruption of six ORFs onSaccharomyces cerevisiae chromosome X: theYJL069c gene of unknown function is essential to cell viability

The short flanking homology PCR strategy (Wach et al., 1994) was used to disrupt six open reading frames (ORFs) on chromosome X of diploid strains (FY1679 and W303) of the yeast Saccharomyces cerevisiae. Two of the six ORFs analysed (YJL069c and YJL066c) display no similarity to known sequences. Three others (YJL065c, YJL068c, and YJL070c) are similar to those respectively encoding the DNA polymerase epsilon subunit c, human esterase D and rat AMP deaminase 1. YJL071w has recently been identified as the ARG2 gene coding for acetylglutamate synthase. Inactivation of the YJL069c gene proved lethal and the yjl071w haploid disruptants were auxotrophic for arginine. For the four other gene inactivations, neither the heterozygous deletion diploids nor the corresponding haploid deletion mutants displayed any special phenotype when grown on rich glycerol or glucose medium or on synthetic minimal medium at three different temperatures, or on media containing compounds interfering with nucleic acid or protein synthesis. Mating and sporulation efficiencies were the same for the viable disruptants as for wild-type cells. The six kanMX4 disruption cassettes were cloned into the pUG7 vector and each of the cognate wild-type genes was inserted into the pRS416 centromeric plasmid. All strains and plasmids have been deposited in the EUROFAN collection (EUROSCARF, K. -D. Entian, Frankfurt, Germany).

Fission yeast cdc24 is a replication factor C- and proliferating cell nuclear antigen-interacting factor essential for S-phase completion.

At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis. The cdc24(+) gene encodes a 501-amino-acid protein with no significant homology to any known proteins. The temperature-sensitive cdc24 mutant is effectively rescued by pcn1(+), rfc1(+) (a fission yeast homologue of RFC1), and hhp1(+), which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively. The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule. In addition, cdc24(+) genetically interacts with the gene encoding the catalytic subunit of DNA polymerase epsilon, which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10. These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.

Schizosaccharomyces pombe cdc20+ encodes DNA polymerase epsilon and is required for chromosomal replication but not for the S phase checkpoint.

In fission yeast both DNA polymerase alpha (pol alpha) and delta (pol delta) are required for DNA chromosomal replication. Here we demonstrate that Schizosaccharomyces pombe cdc20+ encodes the catalytic subunit of DNA polymerase epsilon (pol epsilon) and that this enzyme is also required for DNA replication. Following a shift to the restrictive temperature, cdc20 temperature-sensitive mutant cells block at the onset of DNA replication, suggesting that cdc20+ is required early in S phase very near to the initiation step. In the budding yeast Saccharomyces cerevisiae, it has been reported that in addition to its proposed role in chromosomal replication, DNA pol epsilon (encoded by POL2) also functions directly as an S phase checkpoint sensor [Navas, T. A., Zhou, Z. & Elledge, S. J. (1995) Cell 80, 29-39]. We have investigated whether cdc20+ is required for the checkpoint control operating in fission yeast, and our data indicate that pol epsilon does not have a role as a checkpoint sensor coordinating S phase with mitosis. In contrast, germinating spores disrupted for the gene encoding pol alpha rapidly enter mitosis in the absence of DNA synthesis, suggesting that in the absence of pol alpha, normal coordination between S phase and mitosis is lost. We propose that the checkpoint signal operating in S phase depends on assembly of the replication initiation complex, and that this signal is generated prior to the elongation stage of DNA synthesis.

The ssb-113 allele suppresses the dnaQ49 mutator and alters DNA supercoiling in Escherichia coli.

Mutations in the dnaQ gene, which encodes the proofreading epsilon-subunit of the DNA polymerase III holoenzyme, lead to a mutator phenotype caused by enhanced error rates during DNA replication. In this paper, we studied the influence of ssb mutations on the dnaQ49 mutator, because of the involvement of SSB protein in DNA replication. We found that the ssb-113 mutation suppresses the mutator phenotype of dnaQ49. The suppression effect resulted from an enhanced expression of the dnaQ49 allele as determined by experiments with gene fusions. S1 nuclease analysis revealed that the increased dnaQ expression is based on transcriptional activation of the dnaQP2 promoter. This seems to be the consequence of an increased DNA supercoiling in the ssb-113 mutant, which also influenced further functions that are sensitive to alterations in DNA supercoiling. These results support the hypothesis that the expression of the epsilon-subunit of DNA polymerase III may additionally be modulated by DNA supercoiling, and suggest a possible role for DNA topology in mutagenesis.

Mutator tRNAs are encoded by the Escherichia coli mutator genes mutA and mutC: a novel pathway for mutagenesis.

We have previously described the mutator alleles mutA and mutC, which map at 95 minutes and 42 minutes, respectively, on the Escherichia coli genetic map and which stimulate transversions; the A.T-->T.A and G.C-->T.A substitutions are the most prominent. In this study we show that both mutA and mutC result from changes in the anticodon in one of four copies of the same glycine tRNA, at either the glyV or the glyW locus. This change results in a tRNA that inserts glycine at aspartic acid codons. In view of previous studies of missense suppressor tRNAs, the mistranslation of aspartic acid codons is assumed to occur at approximately 1-2%. We postulate that the mutator tRNA effect is exerted by generating a mutator polymerase and suggest that the epsilon subunit of DNA polymerase, which provides a proofreading function, is the most likely target. The implications of these findings for the contribution of mistranslation to observed spontaneous mutation rates in wild-type strains, as well as other cellular phenomena such as aging, are discussed.

MMS-induced mutagenesis and DNA repair in Escherichia coli dnaQ49: contribution of UmuD' to DNA repair.

dnaQ-encoded epsilon subunit of DNA polymerase III, possesses 3',5' exonuclease (proofreading) activity, and is a fidelity factor of polymerase III holoenzyme. It is assumed that during SOS-induced mutagenesis, UmuD', UmuC, and RecA may suppress DnaQ proofreading activity, and allow for translesional DNA synthesis at the cost of fidelity of replication. In this report SOS-dependent, MMS-induced mutagenesis and DNA repair were tested in E. coli dnaQ49 strains. Bacteria were transformed with various pDNAs harboring compilation of the umuD(D')C genes, and the influence of plasmids on mutagenesis (argE3-->Arg+) and DNA repair was tested. DNA damage and repair were tested in plasmid DNA grown in MMS-treated bacteria and isolated either immediately after MMS treatment, or after starving the cells (MFD conditions) for 30 and 60 min, then nicking activity of Fpg protein on plasmid DNAs was analyzed. It has been found that (i) repair of MMS-induced lesions depends on umuD'C, umuD' (and to much less degree, on umuDC) genes encoded in pDNA; (ii) MMS-induced mutations, in contrast to DNA repair, are highest in the cells transformed with pDNA harboring umuDC, and lowest or zero in cells with plasmids harboring umuD'C. It is postulated that UmuD'C or UmuD' proteins play a role in the repair of damaged DNA and/or in maintenance of DNA integrity. The kinetics of these processes (perhaps due to introducing too many of the lesions) seems to be different in E. coli dnaQ+ and dnaQ cells, and probably this is a reason that (iii) MMS-induced mutations in dnaQ49 strains are not subject to MFD.

The antimutagenic effect of a truncated epsilon subunit of DNA polymerase III in Escherichia coli cells irradiated with UV light.

It has previously been suggested that inhibition of the proofreading 3'-5' exonuclease activity of DNA polymerase may play an important role in generation of UV-induced mutations in Escherichia coli. Our previous work showing that overproduction of epsilon, the proofreading subunit of DNA polymerase III, counteracts the SOS mutagenic response of E. coli seemed to be consistent with this hypothesis. To explore further the nature of the antimutagenic effect of epsilon we constructed plasmid pMK17, which encodes only two of the three highly conserved segments of epsilon--ExoI and ExoII; the third segment, ExoIII, which is essential for 3'-5' exonuclease activity, is deleted. We show that at 40 degrees C, overproduction of the truncated epsilon subunit significantly delays production of M13 phage, suggesting that the protein retains its capacity to bind to DNA. On the other hand, the presence of pMK17 in a trpE65 strain growing at 40 degrees C causes a 10-fold decrease in the frequency of UV-induced Trp+ mutations. This antimutagenic effect of the truncated epsilon is effectively relieved by excess UmuD,C proteins. We also show that the presence of plasmid pIP21, which contains the dnaQ49 allele encoding an epsilon subunit that is defective in proofreading activity, almost completely prevents generation of UV-induced mutations in the trpE65 strain. We propose that the DNA binding ability of free epsilon, rather than its 3'-5' exonuclease activity, affects processing of premutagenic UV-induced lesions, possibly by interfering with the interaction between the UmuC-UmuD'-RecA complex and Pol III holoenzyme. This interaction is probably a necessary condition for translesion synthesis.

Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli.

An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective epsilon subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon. The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene. These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant. We have used this finding as a basis for selection of new plasmid umuC mutants. The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail. In the umuC122::Tn5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC+ plasmid. Interestingly, while the frequency of UV-induced GC-->AT transitions is greatly reduced, the frequency of AT-->TA transversions is not affected. Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC+; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A. We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame. We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.

A strong mutator effect caused by an amino acid change in the alpha subunit of DNA polymerase III of Escherichia coli.

Most potent mutators heretofore detected in Escherichia coli are associated with defects in epsilon subunit of DNA polymerase III, encoded by the dnaQ gene. To elucidate the role of the alpha subunit, the catalytic subunit of the polymerase, in maintaining the high fidelity of DNA replication, we isolated a mutator mutant, the mutation (dnaE173) of which resides on the dnaE gene, encoding the alpha subunit. The dnaE173 mutant was unable to grow in salt-free L broth at temperatures exceeding 44.5 degrees C and exhibited an increased frequency of spontaneous mutations, 1,000 to 10,000-fold the wild type level, at permissive temperatures. The mutator effect of dnaE173 mutation is dominant over the wild type allele. These phenotypes are caused by a single base substitution, resulting in one amino acid change, Glu612 (GAA)----Lys(AAA), in the alpha subunit molecule. DNA polymerase III purified from the dnaE173 mutant contained both alpha and epsilon subunits, in a normal molar ratio. We found no differences between wild type and mutant polymerases in the Vmax, thermolabilities, and salt sensitivities. However, the apparent Km for the substrate nucleotide of the mutant polymerase was 1/6 of that determined with the wild type polymerase. Although the mutant polymerase retained a normal level of 3'----5' exonuclease activity, the proofreading capacity determined by "turnover assay" was significantly lower in the mutant polymerase, as compared with findings in the normal enzyme. It seems likely that the enhanced mutability in the dnaE173 strain results from, at least in part, a defect in the editing function of DNA polymerase III, and further suggests that a portion of the alpha subunit in which the amino acid change resides may be important for the proper setting of the two subunits at the replication fork so as to facilitate efficient editing during the DNA replication.

Requirements for bypass of UV-induced lesions in single-stranded DNA of bacteriophage phi X174 in Salmonella typhimurium.

According to the current model for mutagenic bypass of UV-induced lesions, efficient bypass requires three proteins: activated RecA (RecA*) and either activated UmuD (UmuD') and UmuC or their plasmid-encoded analogues, MucA' and MucB. RecA* aids synthesis of UmuD' and UmuC (and MucA'/MucB) at two levels: by inactivation of the LexA transcriptional repressor of these genes and by cleavage of UmuD (and MucA) to produce the active fragments, UmuD' (MucA'). A third role for RecA is revealed when these two roles are otherwise satisfied in a suitably engineered strain. An often-suggested possible role for RecA in bypass is inhibition of editing by the epsilon subunit of DNA polymerase III. Here, by demonstrating that elimination of epsilon by deletion of its gene, dnaQ, does not relieve the requirement for the third function of RecA, we show that RecA must perform some function other than, or in addition to, inhibition of epsilon. We also show that elimination of epsilon does not relieve the requirement for either Muc protein. Moreover, we observed reactivation of irradiated phi X174 in unirradiated cells expressing MucA' and MucB. This finding makes it unlikely that the additional role of recA involves derepression of an unidentified gene or cleavage of an unidentified protein and makes it more likely that RecA participates directly in bypass.