Error-prone DNA repair consists of replicative filling-in of DNA gaps carrying lesions. We have reconstituted E. coli SOS error-prone repair using purified DNA polymerase III holoenzyme, SSB, RecA, UmuD', a UmuC fusion protein, and a gap lesion plasmid. In the absence of UmuDC, or without SOS induction, replication skips over the lesion, forming mostly one-nucleotide deletions. These cause translational frameshifts that usually inactivate genes. UmuD' and UmuC, in the presence of RecA and SSB, stimulate translesion replication and change its mutagenic specificity such that deletions are prevented and base substitutions are increased. This results in mutagenic but nondetrimental gap repair and provides an effective mechanism for generating genetic variation in bacteria adapting to environmental stress.

When challenged by DNA-damaging agents, Escherichia coli cells respond by inducing the SOS stress response, which leads to an increase in mutation frequency by two mechanisms: translesion replication, a process that causes mutations because of misinsertion opposite the lesions, and an inducible mutator activity, which acts at undamaged sites. Here we report that DNA polymerase V (pol V; UmuC), which previously has been shown to be a lesionbypass DNA polymerase, was highly mutagenic during in vitro gap-filling replication of a gapped plasmid carrying the cro reporter gene. This reaction required, in addition to pol V, UmuD, RecA, and single-stranded DNA (ssDNA)-binding protein. pol V produced point mutations at a frequency of 2.1 ؋ 10 ؊4 per nucleotide (2.1% per cro gene), 41-fold higher than DNA polymerase III holoenzyme. The mutational spectrum of pol V was dominated by transversions (53%), which were formed at a frequency of 1.3 ؋ 10 ؊4 per nucleotide (1.1% per cro gene), 74-fold higher than with pol III holoenzyme. The prevalence of transversions and the protein requirements of this system are similar to those of in vivo untargeted mutagenesis (SOS mutator activity). This finding suggests that replication by pol V, in the presence of UmuD, RecA, and ssDNA-binding protein, is the basis of chromosomal SOS untargeted mutagenesis. The SOS stress response is induced in Escherichia coli by single-stranded DNA (ssDNA) gaps formed when DNA lesions that have escaped repair block replication (1-3). Unable to remove the lesion from such gap͞lesion structures, the cells activate a tolerance response, which involves filling in the DNA gaps without removal of the lesion, thereby restoring genome continuity. Thereafter, a second attempt to eliminate the lesion by DNA repair can be made. Filling in of the gap is done by patching of a homologous DNA segment from the fully replicated sister chromatid via recombinational repair (4, 5) or by translesion replication, which requires the SOS-inducible proteins UmuC, UmuDЈ, and RecA (6, 7). The latter process is mutagenic, because of the miscoding promoted by most DNA lesions. Recently, it was found that UmuC is a DNA polymerase, termed DNA polymerase V (pol V), with a remarkable capability to replicate through DNA lesions that severely block other DNA polymerases (8,9).In addition to this mutagenesis process, which is targeted to DNA lesions, a mutator activity is induced under SOS conditions, which produces mutations in the apparent absence of DNA damage (untargeted mutagenesis) (10-12). Chromosomal untargeted mutagenesis requires the SOS-inducible proteins RecA, UmuDЈ, and UmuC (1, 13, 14), the same proteins that are required for translesion replication. In addition, it exhibits a particular mutational specificity, namely, the selective generation of transversions (14-17). Another pathway of untargeted mutagenesis is observed by transfecting UV-irradiated E. coli cells with unirradiated phage (18). This phage untargeted mutagenesis requires the dinB, uvrA, and polA gene products ...

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MutL␣, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutL␣ and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutL␣ proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutL␣ demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutL␣ activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2. Mismatch repair (MMR)1 helps to protect the genome from replication errors caused by DNA polymerases. Its pivotal role as caretaker of genome stability is exemplified by HNPCC, a hereditary predisposition to colon, rectal, and other cancers associated with germ line mutations in MMR (1).In Escherichia coli, the major players in the pathway are known to the extent that a full in vitro mismatch repair was reconstituted from purified components (2). A MutS homodimer recognizes and binds a mismatch and recruits a MutL homodimer to form an ATP-dependent ternary complex with DNA (3). MutL recruits and activates MutH (4), an endonuclease that introduces a specific nick on hemimethylated GATC sites, thus providing the strand discrimination signal required to ensure repair of the nascent strand. MutL also activates UvrD (5), a helicase that unwinds the duplex DNA from the nick, providing a single strand DNA substrate for an exonuclease. The repair pathway is bidirectional in that repair can initiate from GATC sites located either 5Ј or 3Ј to the mismatch (6). As a result, of the four exonucleases that have been found to participate in the pathway, two have 5Ј to 3Ј polarity, and the others have 3Јto 5Ј polarity (7). The single strand gap formed by the exonuclease is filled in by DNA polymerase III, and the nick is sealed by DNA ligase.The pathway in eukaryotes is less understood than in E. coli (reviewed in Refs. 8 and 9). Instead of one MutS, there are three MutS-like proteins that are involved in mutation avoidance, Msh2, Msh3, and Msh6. Msh2 and Msh6 associate to form MutS␣, the major MutS activity in MMR (reviewed in Ref. 9). Illustrating a similar level of complexity, there are four MutL homologs in mammals: Mlh1, Pms2 (closest to Pms1 in yeast), Pms1, and Mlh3....

The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the ␤ subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate. Addition of the ␤ subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min. When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the ␤ subunit, but it was reduced by 80%. DNA sequence analysis of translesion replication products revealed mostly ؊1 frameshifts. This mutation type is changed to base substitution by the addition of UmuD , UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction. These results indicate that the ␤ subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts.Numerous lesions are formed continuously in DNA (1). Most of these lesions are removed effectively via DNA repair, thus restoring the structural integrity of DNA (1). When DNA lesions that have escaped repair are replicated, they often give rise to mutations caused by the tendency of DNA polymerase to insert an incorrect nucleotide opposite the lesion. This process was termed translesion replication, bypass synthesis, or error-prone repair and is responsible for induced mutagenesis in prokaryotes and in eukaryotes (1-3).In Escherichia coli, replication-blocking lesions, such as the UV light-induced cyclobutyl pyrimidine dimers (2), or abasic sites (4) give rise to mutations in a process that depends on the SOS stress response and requires DNA polymerase III (pol III) and the SOS-regulated proteins RecA, UmuDЈ, and UmuC (1, 2, 5). This led to the hypothesis that pol III alone cannot replicate the ''blocking lesions''; only with the assistance of RecA, UmuDЈ, and UmuC can pol III perform translesion replication through these lesions and give rise to mutations. Studies on the ability of purified pol III holoenzyme to replicate blocking lesions unassisted yielded conflicting results (6-11). Recently, it has been shown that this controversy resulted from the usage of different DNA substrates for the translesion replication assay. Indeed, when a native-like gapped plasmid DNA was used as a substrate, pol III holoenzyme could replicate through a synthetic abasic site (unpublished data). Here, we report that...