The Mutagenesis Protein UmuC Is a DNA Polymerase Activated by UmuD′, RecA, and SSB and Is Specialized for Translesion Replication

Abstract

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...

Stress responses and genetic variation in bacteria

“…Many of these polymerases can replicate damaged DNA, but this ability comes at the cost of frequent mutations on both damaged and undamaged templates. E. coli has two Y-family DNA polymerases, Pol IV, the product of the dinB gene (also called dinP) [6], and Pol V, the product of the umuDC operon [7][8][9]. Both polymerases are repressed by LexA and induced as part of the SOS response [10][11][12].…”

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

“…SOS induces the expression of both DinB and the umuDC operon encoding two polymerases, PolIV and PolV, respectively, for translesion synthesis (TLS). In addition, RecA* stimulates the cleavage of the regulatory subunit UmuD to form a fully functional PolV (UmuD' 2 -UmuC) [12,13], and the RecA-ssDNA filaments are required for both homologous recombination and TLS [14]. This ssDNA-binding activity of RecA has been suggested to induce fork regression forming a chicken-foot structure, or to act as a primer for TLS allowing DDT and replication restart [15], as illustrated in Figure 1.…”

Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

“…Lane 1 in each panel shows a control incubation of the reaction mixture without any DNA polymerase. Other lanes contain the products of reactions performed with the following DNA polymerases: human pol (lanes 2-4); human pol (lanes 5-7); human pol ( lanes 8 -10); E. coli polymerase I (Klenow fragment) (lanes [11][12][13]. For each DNA polymerase, three time points of the reaction are shown.…”