Article

Participation of DNA Polymerase in Replication of Undamaged DNA in Saccharomyces cerevisiae

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Genetics (Impact Factor: 5.96). 10/2009; 184(1):27-42. DOI: 10.1534/genetics.109.107482
Source: PubMed

ABSTRACT

Translesion synthesis DNA polymerases contribute to DNA damage tolerance by mediating replication of damaged templates. Due to the low fidelity of these enzymes, lesion bypass is often mutagenic. We have previously shown that, in Saccharomyces cerevisiae, the contribution of the error-prone DNA polymerase zeta (Polzeta) to replication and mutagenesis is greatly enhanced if the normal replisome is defective due to mutations in replication genes. Here we present evidence that this defective-replisome-induced mutagenesis (DRIM) results from the participation of Polzeta in the copying of undamaged DNA rather than from mutagenic lesion bypass. First, DRIM is not elevated in strains that have a high level of endogenous DNA lesions due to defects in nucleotide excision repair or base excision repair pathways. Second, DRIM remains unchanged when the level of endogenous oxidative DNA damage is decreased by using anaerobic growth conditions. Third, analysis of the spectrum of mutations occurring during DRIM reveals the characteristic error signature seen during replication of undamaged DNA by Polzeta in vitro. These results extend earlier findings in Escherichia coli indicating that Y-family DNA polymerases can contribute to the copying of undamaged DNA. We also show that exposure of wild-type yeast cells to the replication inhibitor hydroxyurea causes a Polzeta-dependent increase in mutagenesis. This suggests that DRIM represents a response to replication impediment per se rather than to specific defects in the replisome components.

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Available from: Matthew R Northam, Apr 15, 2014
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    • "Apart from its function in TLS, polz is known to play a role in homologous recombination (Sharma et al., 2012), nonhomologous end-joining (Covo et al., 2009) and inter-and intrastrand crosslink repair (Enoiu et al., 2012;Hicks et al., 2010). Additionally, yeast polz is able to replicate undamaged DNA (Northam et al., 2010) and shares accessory subunits with polymerase d (Johnson et al., 2012;Makarova et al., 2012) emphasizing its tight relationship with normal DNA replication. Inhibition of REV3 expression in human cells leads to accumulation of DNA double strand breaks (DSB), activation of DNA damage response (DDR) and a reduced fraction of S-phase cells (), which results in increased formation of anaphase bridges and chromosomal breaks/gaps, expression of common fragile sites (CFS), genomic instability (Bhat et al., 2013) and ultimately cell cycle arrest or senescence (). "
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    ABSTRACT: REV3, the catalytic subunit of translesion polymerase zeta (polζ), is commonly associated with DNA damage bypass and repair. Despite sharing accessory subunits with replicative polymerase δ, very little is known about the role of polζ in DNA replication. We previously demonstrated that inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells. To reveal determinants of this sensitivity and obtain insights into the cellular function of REV3, we performed whole human genome RNAi library screens aimed at identification of synthetic lethal interactions with REV3 in A549 lung cancer cells. The top confirmed hit was RRM1, the large subunit of ribonucleotide reductase (RNR), a critical enzyme of de novo nucleotide synthesis. Treatment with the RNR-inhibitor hydroxyurea (HU) synergistically increased the fraction of REV3-deficient cells containing single stranded DNA (ssDNA) as indicated by an increase in replication protein A (RPA). However, this increase was not accompanied by accumulation of the DNA damage marker γH2AX suggesting a role of REV3 in counteracting HU-induced replication stress (RS). Consistent with a role of REV3 in DNA replication, increased RPA staining was confined to HU-treated S-phase cells. Additionally, we found genes related to RS to be significantly enriched among the top hits of the synthetic sickness/lethality (SSL) screen further corroborating the importance of REV3 for DNA replication under conditions of RS.
    Full-text · Article · Dec 2014 · Molecular Oncology
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    • "Apart from its function in TLS, polδ is known to play a role in homologous recombination (Sharma et al., 2012), non-homologous end-joining (Covo et al., 2009) and inter-and intrastrand crosslink repair (Enoiu et al., 2012; Hicks et al., 2010). Additionally, yeast polδ is able to replicate undamaged DNA (Northam et al., 2010) and shares accessory subunits with polymerase δ (Johnson et al., 2012; Makarova et al., 2012) emphasizing its tight relationship with normal DNA replication. Inhibition of REV3 expression in human cells leads to accumulation of DNA double strand breaks (DSB), activation of DNA damage response (DDR) and a reduced fraction of S-phase cells (Knobel et al., 2011), which results in increased formation of anaphase bridges and chromosomal breaks/gaps, expression of common fragile sites (CFS), genomic instability (Bhat et al., 2013) and ultimately cell cycle arrest or senescence (Knobel et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: REV3, the catalytic subunit of translesion polymerase zeta (polζ), is commonly associated with DNA damage bypass and repair. Despite sharing accessory subunits with replicative polymerase δ, very little is known about the role of polζ in DNA replication. We previously demonstrated that inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells. To reveal determinants of this sensitivity and obtain insights into the cellular function of REV3, we performed whole human genome RNAi library screens aimed at identification of synthetic lethal interactions with REV3 in A549 lung cancer cells. The top confirmed hit was RRM1, the large subunit of ribonucleotide reductase (RNR), a critical enzyme of de novo nucleotide synthesis. Treatment with the RNR-inhibitor hydroxyurea (HU) synergistically increased the fraction of REV3-deficient cells containing single stranded DNA (ssDNA) as indicated by an increase in replication protein A (RPA). However, this increase was not accompanied by accumulation of the DNA damage marker γH2AX suggesting a role of REV3 in counteracting HU-induced replication stress (RS). Consistent with a role of REV3 in DNA replication, increased RPA staining was confined to HU-treated S-phase cells. Additionally, we found genes related to RS to be significantly enriched among the top hits of the synthetic sickness/lethality (SSL) screen further corroborating the importance of REV3 for DNA replication under conditions of RS.
    Full-text · Article · Jul 2014 · Molecular Oncology
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    • "In yeast, deletion of REV3 is not lethal but causes growth retardation in strains with elevated levels of abasic sites [26]. Loss of the catalytic subunit of pol ␨ or Rev1 results in elevated rates of large deletions [24] [25] and gross chromosomal abnormalities [27]. Therefore, while error-prone TLS is etiologic in most environmentally induced cancers, its absence can also contribute to genome instability and cancer [13] [28] [29]. "
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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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