Zhong, X. et al. The fidelity of DNA synthesis by yeast DNA polymerase alone and with accessory proteins. Nucleic Acids Res. 34, 4731-4742

Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA.
Nucleic Acids Research (Impact Factor: 9.11). 02/2006; 34(17):4731-42. DOI: 10.1093/nar/gkl465
Source: PubMed


DNA polymerase zeta (pol ζ) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced
mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol ζ alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol
ζ. Pol ζ is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity
of pol ζ is substantially lower than that of homologous B family pols α, δ and ɛ. Pol ζ is particularly error prone for substitutions
in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented
in comparison with other polymerases. The unique error specificity of pol ζ in vitro is consistent with Pol ζ-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates
that pol ζ contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending

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Available from: Peter M J Burgers, Jan 29, 2014
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    • "Pol ␨ is responsible for most induced point mutations and roughly half of spontaneous mutations [9] [21]. It synthesizes DNA in vitro with low fidelity and produces a characteristic mutational signature [22], found in mutation spectra in vivo [2] [23] [24]. Part of the signature is attributed to template switches [25]. "
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    ABSTRACT: Unrepaired DNA lesions often stall replicative DNA polymerases and are bypassed by translesion synthesis (TLS) to prevent replication fork collapse. Mechanisms of TLS are lesion- and species-specific, with a prominent role of specialized DNA polymerases with relaxed active sites. After nucleotide(s) are incorporated across from the altered base(s), the aberrant primer termini are typically extended by DNA polymerase ζ (pol ζ). As a result, pol ζ is responsible for most DNA damage-induced mutations. The mechanisms of sequential DNA polymerase switches in vivo remain unclear. The major replicative DNA polymerase δ (pol δ) shares two accessory subunits, called Pol31/Pol32 in yeast, with pol ζ. Inclusion of Pol31/Pol32 in the pol δ/pol ζ holoenzymes requires a [4Fe-4S] cluster in C-termini of the catalytic subunits. Disruption of this cluster in Pol ζ or deletion of POL32 attenuates induced mutagenesis. Here we describe a novel mutation affecting the catalytic subunit of pol ζ, rev3ΔC, which provides insight into the regulation of pol switches. Strains with Rev3ΔC, lacking the entire C-terminal domain and therefore the platform for Pol31/Pol32 binding, are partially proficient in Pol32-dependent UV-induced mutagenesis. This suggests an additional role of Pol32 in TLS, beyond being a pol ζ subunit, related to pol δ. In search for members of this regulatory pathway, we examined the effects of Maintenance of Genome Stability 1 (Mgs1) protein on mutagenesis in the absence of Rev3-Pol31/Pol32 interaction. Mgs1 may compete with Pol32 for binding to PCNA. Mgs1 overproduction suppresses induced mutagenesis, but had no effect on UV-mutagenesis in the rev3ΔC strain, suggesting that Mgs1 exerts its inhibitory effect by acting specifically on Pol32 bound to pol ζ. The evidence for differential regulation of Pol32 in pol δ and pol ζ emphasizes the complexity of polymerase switches.
    Full-text · Article · May 2014 · DNA repair
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    • "The strong reduction in the rate of complex mutations in the pol3-Y708A rev1-cd strain, however, suggests that the C insertion by Rev1 is somehow required for their generation by Pol ζ in vivo. This observation was in apparent disagreement with the fact that purified Pol ζ is perfectly capable of making the complex mutations without the help of Rev1 in vitro (7). In the following sections, we propose and vigorously test a model that resolved this controversy and provided insight into the mechanism of replication-associated Pol ζ- and Rev1-dependent mutagenesis. "
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    ABSTRACT: DNA polymerase ζ (Pol ζ) and Rev1 are key players in translesion DNA synthesis. The error-prone Pol ζ can also participate in replication of undamaged DNA when the normal replisome is impaired. Here we define the nature of the replication disturbances that trigger the recruitment of error-prone polymerases in the absence of DNA damage and describe the specific roles of Rev1 and Pol ζ in handling these disturbances. We show that Pol ζ/Rev1-dependent mutations occur at sites of replication stalling at short repeated sequences capable of forming hairpin structures. The Rev1 deoxycytidyl transferase can take over the stalled replicative polymerase and incorporate an additional ‘C’ at the hairpin base. Full hairpin bypass often involves template-switching DNA synthesis, subsequent realignment generating multiply mismatched primer termini and extension of these termini by Pol ζ. The postreplicative pathway dependent on polyubiquitylation of proliferating cell nuclear antigen provides a backup mechanism for accurate bypass of these sequences that is primarily used when the Pol ζ/Rev1-dependent pathway is inactive. The results emphasize the pivotal role of noncanonical DNA structures in mutagenesis and reveal the long-sought-after mechanism of complex mutations that represent a unique signature of Pol ζ.
    Full-text · Article · Sep 2013 · Nucleic Acids Research
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    • "This is because TLS polymerases can contain relatively large active sites that accommodate noncanonical bases (e.g., [15] [16]) and lack the proofreading activity of the higher fidelity replicative polymerases (reviewed in [17] [18]). As a result, TLS polymerases are considerably more errorprone , even when copying undamaged DNA templates (e.g., [19] [20]). Thus, cells have evolved these low fidelity polymerases as a means to bypass genomic lesions that would otherwise block replication, but at the cost of possible mutation fixation [12] [13] [14]. "
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    ABSTRACT: Abasic sites in genomic DNA can be a significant source of mutagenesis in biological systems, including human cancers. Such mutagenesis requires translesion DNA synthesis (TLS) bypass of the abasic site by specialized DNA polymerases. The abasic site bypass specificity of TLS proteins had been studied by multiple means in vivo and in vitro, although the generality of the conclusions reached have been uncertain. Here, we introduce a set of yeast reporter strains for investigating the in vivo specificity of abasic site bypass at numerous random positions within chromosomal DNA. When shifted to 37°C, these strains underwent telomere uncapping and resection that exposed reporter genes within a long 3' ssDNA overhang. Human APOBEC3G cytosine deaminase was expressed to create uracils in ssDNA, which were excised by uracil-DNA N-glycosylase. During repair synthesis, error-prone TLS bypassed the resulting abasic sites. Because of APOBEC3G's strict motif specificity and the restriction of abasic site formation to only one DNA strand, this system provides complete information about the location of abasic sites that led to mutations. We recapitulated previous findings on the roles of REV1 and REV3. Further, we found that sequence context can strongly influence the relative frequency of A or C insertion. We also found that deletion of Pol32, a non-essential common subunit of Pols δ and ζ, resulted in residual low-frequency C insertion dependent on Rev1 catalysis. We summarize our results in a detailed model of the interplay between TLS components leading to error-prone bypass of abasic sites. Our results underscore the utility of this system for studying TLS bypass of many types of lesions within genomic DNA.
    Full-text · Article · Aug 2013 · DNA repair
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