The Architecture of Yeast DNA Polymerase ζ

Gomez‐Llorente, Yacob; Malik, Radhika; Jain, Rinku; Choudhury, Jayati Roy; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Ubarretxena-Belandia, Iban; Aggarwal, Aneel K.

doi:10.1016/j.celrep.2013.08.046

Cited by 34 publications

(52 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Consistently, in S. cerevisiae, the levels of both Rev1 protein and its phosphorylation status increase during the cell cycle, reaching a strong peak at the G2/M phase [117]. The possibility that Rev1 and Pol act post-replicatively at ssDNA gaps is also consistent with the finding that, in S. cerevisiae and mammals, Rev1/Pol can interact with subunits of Pol ␦ [118][119][120][121][122][123], the replicative polymerase that performs lagging strand DNA synthesis, at least in S. cerevisiae [124,125]. Indeed, PolD2 and PolD3 are important for translesion synthesis at helix-distorting lesions [126,127] and spontaneous mutagenesis in vivo [128][129][130].…”

Section: Topological Aspects Of Rev1 and Pol Functionsupporting

confidence: 73%

Roles of mutagenic translesion synthesis in mammalian genome stability, health and disease

2015

View full text Add to dashboard Cite

Section: Topological Aspects Of Rev1 and Pol Functionsupporting

confidence: 73%

Roles of mutagenic translesion synthesis in mammalian genome stability, health and disease

2015

View full text Add to dashboard Cite

“…In yeast, Pol31 directly interacts with the CTD of Pol ζ and Pol δ, and Pol32 interacts only with Pol31 (38). However, the recent EM study of yeast Pol ζ4 placed Pol32 proximal to Rev7 (46). Potential interactions between human PolD3 (yeast Pol32 homolog) and Rev7 may explain our observation that PolD3 remains a component of Pol ζ4, even when PolD3 is in molar excess of PolD2 (Fig.…”

Section: Discussionmentioning

confidence: 78%

Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass

Lee

Gregory

Yang

2014

Proc. Natl. Acad. Sci. U.S.A.

158

189

View full text Add to dashboard Cite

DNA polymerase ζ (Pol ζ) is a eukaryotic B-family DNA polymerase that specializes in translesion synthesis and is essential for normal embryogenesis. At a minimum, Pol ζ consists of a catalytic subunit Rev3 and an accessory subunit Rev7. Mammalian Rev3 contains >3,000 residues and is twice as large as the yeast homolog. To date, no vertebrate Pol ζ has been purified for biochemical characterization. Here we report purification of a series of human Rev3 deletion constructs expressed in HEK293 cells and identification of a minimally catalytically active human Pol ζ variant. With a tagged form of an active Pol ζ variant, we isolated two additional accessory subunits of human Pol ζ, PolD2 and PolD3. The purified foursubunit Pol ζ4 (Rev3-Rev7-PolD2-PolD3) is much more efficient and more processive at bypassing a 1,2-intrastrand d(GpG)-cisplatin cross-link than the two-subunit Pol ζ2 (Rev3-Rev7). We show that complete bypass of cisplatin lesions requires Pol η to insert dCTP opposite the 3′ guanine and Pol ζ4 to extend the primers., composed of the catalytic Rev3 and accessary Rev7 subunits, is an error-prone DNA translesion polymerase that causes both spontaneous and DNA damage-induced mutagenesis (1, 2). More than two-thirds of the 1,504 residues in yeast Rev3 share sequence homology with all B-family DNA polymerases, including Pols α, δ, and e, which are responsible for the bulk of high-fidelity genomic replication in eukaryotes (3). Unlike the typical B-family polymerases, Pol ζ lacks an intrinsic 3′-5′ exonuclease activity and thus has no proofreading function (2). Human homologs of REV3 (REV3L) and REV7 (MAD2L2; hereafter referred to as REV7) genes were identified shortly after yeast Pol ζ was characterized. Human Rev3 contains 3,130 residues and is twice as large as the yeast counterpart (4). Human and yeast Rev7 are homologous (5) and bear sequence similarity to the mitotic checkpoint proteins Mad2 (6). Unlike Saccharomyces cerevisiae REV3 and REV7 genes, which are nonessential and whose knockout leads only to a decreased rate of damage-induced mutagenesis (7, 8), Rev3l knockout in mice is embryonic-lethal (9), and mouse Rev3l −/− embryonic stem cells are not viable (10, 11). Human and mouse cell cultures obtained from conditional Rev3l knockout show genome instability and growth defects without an external challenge of DNA damage (12)(13)(14). DNA pol ζ is apparently essential for normal cell proliferation and embryogenesis in mammals.Translesion synthesis (TLS) and DNA-damage-induced mutagenesis are the best-characterized functions of Pol ζ. Absence of the yeast REV3 gene leads to sensitivity to UV light and intrastrand and interstrand cross-linking agents (2, 15). DNA Pol ζ has been shown to induce multiple base substitutions as well as more complex mutations in yeast (7,16,17) and may contribute to hypermutation in Ig genes in mammals (18,19). The TLS function of DNA Pol ζ has been implicated in its role of mediating resistance to platinum-based chemotherapies (20)(21)(22). Owing to the conservation of B-f...

show abstract

“…Yeast pol ζ can also stably exist as a four-subunit enzyme, containing the catalytic subunit Rev3, accessory subunit Rev7, and Pol31/Pol32 [34–36]. In this complex, Pol32 binds to Pol31, and Pol31 binds to the CTD of catalytic subunit Pol3 [37–39]. The existence of shared subunits between replicative and TLS pols was the basis for the proposal of an additional mechanism of switching between pol δ and pol ζ through an exchange of the catalytic subunits on Pol31/Pol32 bound to PCNA [32].…”

Section: Introductionmentioning

confidence: 99%

“…It is believed that based on the structure of another B-family member pol α and a low resolution EM structure of pol ζ, that both Pol3 and Rev3 contain a CTD attached by a flexible linker [39,41]. Both polymerases contain a FeS cluster in this domain [42], which is required for binding to Pol31/Pol32 [32,34,35].…”

Section: Introductionmentioning

confidence: 99%

A novel variant of DNA polymerase ζ, Rev3ΔC, highlights differential regulation of Pol32 as a subunit of polymerase δ versus ζ in Saccharomyces cerevisiae

et al. 2014

View full text Add to dashboard Cite

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.

show abstract

The Architecture of Yeast DNA Polymerase ζ

Cited by 34 publications

References 36 publications

Roles of mutagenic translesion synthesis in mammalian genome stability, health and disease

Roles of mutagenic translesion synthesis in mammalian genome stability, health and disease

Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass

A novel variant of DNA polymerase ζ, Rev3ΔC, highlights differential regulation of Pol32 as a subunit of polymerase δ versus ζ in Saccharomyces cerevisiae

Contact Info

Product

Resources

About