Replication of DNA lesions leads to the formation of mutations. In Escherichia coli this process is regulated by the SOS stress response, and requires the mutagenesis proteins UmuC and UmuD. Analysis of translesion replication using a recently reconstituted in vitro system (Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199) revealed that lesion bypass occurred with a UmuC fusion protein, UmuD, RecA, and SSB in the absence of added DNA polymerase. Further analysis revealed that UmuC was a DNA polymerase (E. coli DNA polymerase V), with a weak polymerizing activity. Upon addition of UmuD, RecA, and SSB, the UmuC DNA polymerase was greatly activated, and replicated a synthetic abasic site with great efficiency (45% bypass in 6 min), 10 -100-fold higher than E. coli DNA polymerases I, II, or III holoenzyme. Analysis of bypass products revealed insertion of primarily dAMP (69%), and to a lesser degree dGMP (31%) opposite the abasic site. The UmuC104 mutant protein was defective both in lesion bypass and in DNA synthesis. These results indicate that UmuC is a UmuD-, RecA-, and SSB-activated DNA polymerase, which is specialized for lesion bypass. UmuC is a member of a new family of DNA polymerases which are specialized for lesion bypass, and include the yeast RAD30 and the human XP-V genes, encoding DNA polymerase .Mutagenesis caused by UV light and by many other DNA damaging agents in Escherichia coli is under control of the SOS response, a highly regulated stress response, which functions to increase cell survival under adverse environmental conditions that cause DNA damage (1). Genetic analysis has uncovered four genes, whose products are required for SOS mutagenesis. Two of these, DNA polymerase III (pol-III) 1 and RecA, participate also in replication and recombination, respectively.The other two, UmuD and UmuC, are specifically required for the mutagenic reaction. It was found that UmuD is processed into a shorter form, UmuDЈ, which is the form active in SOS mutagenesis (reviewed in Ref.2).Based on in vivo and in vitro data, UmuDЈ and UmuC were thought to be accessory proteins, which assist DNA polymerase III in replicating DNA lesions which usually block replication (2-5). According to this mechanism, the mutations occur by misinsertion opposite the DNA lesion by the DNA polymerase, a result of the miscoding nature of most DNA lesions. Recently SOS mutagenesis was reconstituted with purified components in two laboratories (6, 7). The results, which confirmed an earlier study (4), provided strong biochemical evidence that SOS mutagenesis occurs by replication through DNA lesions, in a reaction which depends on UmuC, UmuDЈ, RecA and SSB. Moreover, it was shown that there is a qualitative difference in the specificity of bypass when translesion replication was compared in the absence or presence of SOS proteins. DNA polymerase III holoenzyme bypassed an abasic site via a misalignment mechanism, resulting in skipping over the lesion, and the formation of Ϫ1 frameshifts (7,8). In contrast, in the p...

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOSregulated proteins UmuD and RecA and the singlestrand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATP␥S, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.Genomic DNA is afflicted by numerous lesions that might interfere with its propagation and with gene expression (1). Most of these lesions, which are usually base modifications, are repaired by cellular DNA repair mechanisms (1). When the replication fork encounters a blocking DNA lesion that has escaped repair, replication stops forming a ssDNA 1 region in DNA (2). In Escherichia coli at least two mechanisms, which are regulated by the SOS response (3), act to repair the gap by converting the ssDNA region into a dsDNA region without actually removing the damaged nucleotide. Recombinational repair patches the gap with a complementary DNA segment from the fully replicated sister chromatid (4, 5), whereas translesion replication fills in the gap by DNA synthesis. This pathway, also termed lesion bypass or error-prone repair, is mutagenic, because DNA lesions often cause misincorporation by DNA polymerases, leading to the formation of mutations (2, 6).The in vitro reconstitution of SOS translesion replication with purified components (7-9) established that SOS-targeted mutagenesis occurs by replication through DNA lesions by DNA polymerase V (UmuC) 2 in the presence of UmuDЈ, RecA, and SSB (10, 11). Pol V effectively bypasses a synthetic abasic site (10, 11), a cyclobutyl TT dimer and a 6-4 TT adduct (12), leading to targeted mutations. When replicating an undamaged DNA template pol V is highly mutagenic and forms preferentially purine-purine and pyrimidine-pyrimidine mismatches, resulting in transversion mutations (13). These activities of pol V are responsible for SOS mutagenesis targeted to DNA lesions and for untargeted mutagenesis, which occurs in undamaged DNA regions.Proteins similar to UmuC are widespread from E. coli...

Translesion DNA synthesis (TLS) by DNA polymerase V (polV) in Escherichia coli involves accessory proteins, including RecA and single-stranded DNA-binding protein (SSB). To elucidate the role of SSB in TLS we used an in vitro exonuclease protection assay and found that SSB increases the accessibility of 3 primer termini located at abasic sites in RecA-coated gapped DNA. The mutant SSB-113 protein, which is defective in protein-protein interactions, but not in DNA binding, was as effective as wild-type SSB in increasing primer termini accessibility, but deficient in supporting polV-catalyzed TLS. Consistently, the heterologous SSB proteins gp32, encoded by phage T4, and ICP8, encoded by herpes simplex virus 1, could replace E. coli SSB in the TLS reaction, albeit with lower efficiency. Immunoprecipitation experiments indicated that polV directly interacts with SSB and that this interaction is disrupted by the SSB-113 mutation. Taken together our results suggest that SSB functions to recruit polV to primer termini on RecA-coated DNA, operating by two mechanisms: 1) increasing the accessibility of 3 primer termini caused by binding of SSB to DNA and 2) a direct SSB-polV interaction mediated by the C terminus of SSB.Translesion DNA synthesis is a DNA damage tolerance mechanism in which replication blocks caused by DNA lesions are relieved by specialized DNA polymerases proficient in synthesizing DNA across lesions (1). In Escherichia coli this reaction is catalyzed primarily by the Y family DNA polymerases encoded by the umuDC operon (DNA polymerase V (polV) 2 ) and the dinB gene (DNA polymerase IV (polIV)) (2-5). The in vitro reconstitution of TLS with purified proteins indicated that both RecA and single-stranded DNA-binding protein (SSB) are required for polV-catalyzed TLS (6 -9). RecA, the main recombinase in E. coli, is also the main activator of the SOS stress response via its ability to promote the autocleavage of the LexA repressor, which is the negative regulator of all SOS genes. RecA has two additional roles in TLS: (a) It activates UmuD by promoting its autocleavage to UmuDЈ, which then binds to UmuC to form the UmuDЈ 2 C complex polV (1). (b) A direct role in TLS which involves the recruitment of UmuDЈ to DNA (10). RecA promotes these activities in its DNA-bound form, which consists of a nucleoprotein filament formed by its cooperative binding to ssDNA (11).SSB is an essential protein in E. coli, involved in replication, recombination and repair. Functional homologs of SSB are present in all organisms, and their importance is well illustrated by the occurrence of viral SSBs (12-15). E. coli SSB is a 75kDa tetramer composed of four identical subunits. It binds ssDNA strongly via a ssDNA-binding domain located at its N terminus, and interacts with a variety of proteins via its C terminus (15)(16)(17)(18)(19)(20). A detailed kinetic analysis showed that SSB had a dramatic effect on TLS by polV across an abasic site (21), however, under some conditions SSB was found to be stimulatory rather than essential f...

Mutations in the human genome are clustered in hot-spot regions, suggesting that some sequences are more prone to accumulate mutations than others. These regions are therefore more likely to lead to the development of cancer. Several pathways leading to the creation of mutations may be influenced by the DNA sequence, including sensitivity to DNA damaging agents, and repair mechanisms. We have analyzed sequence context effects on translesion replication, the error-prone repair of single-stranded DNA regions carrying lesions. By using synthetic oligonucleotides containing systematic variations of sequences flanking a synthetic abasic site, we show that translesion replication by the repair polymerase DNA polymerase beta is stimulated to a moderate extent by low stacking levels of the template nucleotides downstream of the lesion, combined with homopolymeric runs flanking the lesion both upstream and downstream. A strong stimulation of translesion replication by DNA polymerase beta was seen when fork-like flap structures were introduced into the DNA substrate downstream of the lesion. Unlike for gapped substrates, this stimulation was independent of the presence of a phosphate group at the 5' terminus of the flap. These results suggest that DNA polymerase beta may participate in cellular DNA transactions involving higher order structures. The significance of these results for in vivo translesion replication is discussed.

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