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


ABSTRACT: DNA polymerase ? (Pol ?) is a eukaryotic B-family DNA polymerase that specializes in translesion synthesis and is essential for normal embryogenesis. At a minimum, Pol ? 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 ? 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 ? variant. With a tagged form of an active Pol ? variant, we isolated two additional accessory subunits of human Pol ?, PolD2 and PolD3. The purified four-subunit Pol ?4 (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 ?2 (Rev3-Rev7). We show that complete bypass of cisplatin lesions requires Pol ? to insert dCTP opposite the 3' guanine and Pol ?4 to extend the primers.

SUBMITTER: Lee YS 

PROVIDER: S-EPMC3939873 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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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 Young-Sam YS   Gregory Mark T MT   Yang Wei W  

Proceedings of the National Academy of Sciences of the United States of America 20140121 8


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 cel  ...[more]

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