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...

The human 3-methyladenine DNA glycosylase (AAG) recognizes and excises a broad range of purines damaged by alkylation and oxidative damage, including 3-methyladenine, 7-methylguanine, hypoxanthine (Hx), and 1,N 6 -ethenoadenine (εA). The crystal structures of AAG bound to εA have provided insights into the structural basis for substrate recognition, base excision, and exclusion of normal purines and pyrimidines from its substrate recognition pocket. In the present study, we explored the substrate specificity of full-length and truncated Δ80AAG on a library of oligonucleotides containing structurally diverse base modifications. Substrate binding and base excision kinetics of AAG with 13 damaged oligonucleotides were examined. We found that AAG bound to a wide variety of purine and pyrimidine lesions, but excised only few of them. Singleturnover excision kinetics showed that in addition to the well-known εA and Hx substrates, 1-methylguanine (m1G) was also excised efficiently by AAG. Thus, along with εA and ethanoadenine (EA), m1G is another substrate that is shared between AAG and the direct repair protein AlkB. In addition, we found that both the full-length and truncated AAG excised 1,N 2 -ethenoguanine (1,N 2 -εG), albeit weakly, from duplex DNA. Uracil was excised from both single-and double-stranded DNA, but only by the full-length AAG, indicating that the N-terminus of AAG may influence glycosylase activity for some substrates. Although AAG has been primarily shown to act on doublestranded DNA, AAG excised both εA and Hx from single-stranded DNA, suggesting the possible significance of repair of these frequent lesions in single-stranded DNA transiently generated during replication and transcription.DNA damaging agents are ubiquitous and cellular DNA is constantly attacked by a variety of endogenous and exogenous DNA damaging agents. DNA can be deaminated spontaneously or alkylated by endogenous intracellular sources and by exogenous environmental agents. Such damages can interfere with DNA replication and transcription, and may be mutagenic or † This work was supported by NIH grants (ES05355, CA75576, CA55042, ES02109, T32-ES007020, CA80024, and CA26731) and NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 10. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript cytotoxic to the cell. During evolution, multiple DNA repair pathways have evolved to maintain the integrity of DNA in all organisms. Among other pathways, single base aberrations can be repaired by the base excision repair (BER) pathway. BER is initiated by DNA glycosylases that recognize the damaged base in the genome, followed by hydrolysis of the N-glycosylic bond, resulting in the release of the damaged base and the generation of an abasic site. The abasic site is further processed by an AP endonuclease or AP lyase, resulting in a strand break. After trimming of the DNA ends, DNA is resynthesized by a DNA polymerase and a DNA ligase seals the nick to restore undamaged ...

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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