Structural Basis of Rev1-mediated Assembly of a Quaternary Vertebrate Translesion Polymerase Complex Consisting of Rev1, Heterodimeric Polymerase (Pol) ζ, and Pol κ

Wojtaszek, Jessica L.; Lee, Chul‐Jin; D’Souza, Sanjay; Minesinger, Brenda K.; Kim, Hyungjin; D’Andrea, Alan D.; Walker, Graham C.; Zhou, Pei

doi:10.1074/jbc.m112.394841

Cited by 105 publications

(150 citation statements)

References 47 publications

(61 reference statements)

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“…Band x was identified to be α and β tubulins. The other protein band (band y) pulled down with or without the Rev3 CTD was ∼75 kDa and is too small to be Rev1, which is known to associate weakly with Rev7 (39)(40)(41)(42). Based on our prior experience with protein expression in HEK293 cells, band y is likely a protein chaperone.…”

Section: Resultsmentioning

confidence: 99%

“…Our pull-down experiments using whole-cell lysates of HEK293 cells, however, were conducted without the assumption of PolD2 and PolD3 involvement. Only PolD2 and PolD3 copurified with Rev3 (TR4-2) and Rev7 after stringent washing with a 1.0 M NaCl solution, and Rev1, a known cofactor of Pol ζ via interactions with Rev7 (41,42,45), was not detected (Fig. 2B).…”

Section: Discussionmentioning

confidence: 99%

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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.

161

189

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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.

161

189

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“…polymerase, and it is essential for ICL repair (33), where it presumably engages by coordinating the REV7 subunit of pol (34,35).…”

Section: Discussionmentioning

confidence: 99%

A Small Molecule Inhibitor of Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) Inhibits Repair of Interstrand DNA Cross-link, Enhances DNA Double Strand Break, and Sensitizes Cancer Cells to Cisplatin

Inoue

Kikuchi

Hishiki

et al. 2014

Journal of Biological Chemistry

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Background: Lys-164-monoubiquitinated PCNA is essential for interstrand DNA cross-link (ICL) repair. Results: A small molecule, T2AA, bi-molecularly binds to PCNA at a PIP-box cavity and close to Lys-164. T2AA inhibited monoubiquitinated PCNA interactions and ICL repair and enhanced DNA double strand breaks. Conclusion: An inhibitor of monoubiquitinated PCNA inhibits ICL repair. Significance: Inhibition of monoubiquitinated PCNA could improve chemotherapeutic efficacy.

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“…We generated KI mice instead of KO mice because Polk interacts with other proteins, such as REV1 and PCNA [23,[36][37][38]. REV1 interacts with Pol eta, Pol iota, and REV3L (a catalytic subunit of Pol zeta) [39], and PCNA interacts with a number of proteins involved in DNA transactions [40]. Therefore, a simple KO or knockdown of Polk might possibly modulate the functions of other proteins, thereby obscuring the intrinsic roles of Polk, which depend on a catalytic activity as a DNA polymerase and on protein-protein cross-talks with other DNA replication components to maintain the genome stability.…”

Section: Introductionmentioning

confidence: 99%

In vivo evidence that DNA polymerase kappa is responsible for error-free bypass across DNA cross-links induced by mitomycin C

Takeiri¹,

Wada²,

Motoyama³

et al. 2014

DNA Repair

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Structural Basis of Rev1-mediated Assembly of a Quaternary Vertebrate Translesion Polymerase Complex Consisting of Rev1, Heterodimeric Polymerase (Pol) ζ, and Pol κ

Cited by 105 publications

References 47 publications

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

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

A Small Molecule Inhibitor of Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) Inhibits Repair of Interstrand DNA Cross-link, Enhances DNA Double Strand Break, and Sensitizes Cancer Cells to Cisplatin

In vivo evidence that DNA polymerase kappa is responsible for error-free bypass across DNA cross-links induced by mitomycin C

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