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The POLD3 subunit of DNA polymerase ? can promote translesion synthesis independently of DNA polymerase ?.

ABSTRACT: The replicative DNA polymerase Pol? consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ?. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Pol?, suggesting that POLD3 makes a significant contribution to TLS independently of Pol? in DT40 cells. Indeed, combining pol?, pol? and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Pol? holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Pol? to complete abasic site bypass independently of canonical TLS polymerases.

SUBMITTER: Hirota K

PROVIDER: S-EPMC4330384 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ.

Hirota Kouji K Yoshikiyo Kazunori K Guilbaud Guillaume G Tsurimoto Toshiki T Murai Junko J Tsuda Masataka M Phillips Lara G LG Narita Takeo T Nishihara Kana K Kobayashi Kaori K Yamada Kouich K Nakamura Jun J Pommier Yves Y Lehmann Alan A Sale Julian E JE Takeda Shunichi S

Nucleic acids research 20150127 3

The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with nor ...[more]

PMID: 25628356

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Project description:The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5'-2-deoxyribose-5-phosphate lyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or an 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated -1 or -2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of -2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of -1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non-homologous end joining pathway.

Dataset Information

The POLD3 subunit of DNA polymerase ? can promote translesion synthesis independently of DNA polymerase ?.

Publications

The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ.

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