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

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 lar...

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Published in:Proceedings of the National Academy of Sciences - PNAS 2014-02, Vol.111 (8), p.2954-2959
Main Authors: Lee, Young-Sam, Gregory, Mark T., Yang, Wei
Format: Article
Language:English
Subjects:
Biological Sciences
Catalytic activity
Chromatography, Gel
Chromatography, Ion Exchange
Cisplatin - metabolism
DNA
DNA - biosynthesis
DNA Polymerase III - metabolism
DNA Primers - genetics
DNA Repair - physiology
DNA-Binding Proteins - metabolism
DNA-Directed DNA Polymerase - isolation & purification
DNA-Directed DNA Polymerase - metabolism
Fluorescein
Genetic mutation
HEK293 Cells
Holoenzymes - isolation & purification
Holoenzymes - metabolism
Humans
Lesions
Mad2 Proteins - metabolism
Nucleic acids
Nucleotides
Proteins
Saccharomyces cerevisiae
Yeasts
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cited_by cdi_FETCH-LOGICAL-c4101-85b1bd6cd5e0246188cafcac8475d918d1e29d371728f6381573e71239002ba53
cites cdi_FETCH-LOGICAL-c4101-85b1bd6cd5e0246188cafcac8475d918d1e29d371728f6381573e71239002ba53
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container_title Proceedings of the National Academy of Sciences - PNAS
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creator Lee, Young-Sam
Gregory, Mark T.
Yang, Wei
description 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 four-subunit 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.
doi_str_mv 10.1073/pnas.1324001111
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Gregory, Mark T. ; Yang, Wei</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4101-85b1bd6cd5e0246188cafcac8475d918d1e29d371728f6381573e71239002ba53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Biological Sciences</topic><topic>Catalytic activity</topic><topic>Chromatography, Gel</topic><topic>Chromatography, Ion Exchange</topic><topic>Cisplatin - metabolism</topic><topic>DNA</topic><topic>DNA - biosynthesis</topic><topic>DNA Polymerase III - metabolism</topic><topic>DNA Primers - genetics</topic><topic>DNA Repair - physiology</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>DNA-Directed DNA Polymerase - isolation &amp; purification</topic><topic>DNA-Directed DNA Polymerase - metabolism</topic><topic>Fluorescein</topic><topic>Genetic mutation</topic><topic>HEK293 Cells</topic><topic>Holoenzymes - isolation &amp; purification</topic><topic>Holoenzymes - metabolism</topic><topic>Humans</topic><topic>Lesions</topic><topic>Mad2 Proteins - metabolism</topic><topic>Nucleic acids</topic><topic>Nucleotides</topic><topic>Proteins</topic><topic>Saccharomyces cerevisiae</topic><topic>Yeasts</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lee, Young-Sam</creatorcontrib><creatorcontrib>Gregory, Mark T.</creatorcontrib><creatorcontrib>Yang, Wei</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Nucleic Acids Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lee, Young-Sam</au><au>Gregory, Mark T.</au><au>Yang, Wei</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>2014-02-25</date><risdate>2014</risdate><volume>111</volume><issue>8</issue><spage>2954</spage><epage>2959</epage><pages>2954-2959</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><notes>http://dx.doi.org/10.1073/pnas.1324001111</notes><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><notes>Contributed by Wei Yang, December 27, 2013 (sent for review December 5, 2013)</notes><notes>Author contributions: Y.-S.L. and W.Y. designed research; Y.-S.L. and M.T.G. performed research; Y.-S.L., M.T.G., and W.Y. analyzed data; and Y.-S.L. and W.Y. wrote the paper.</notes><notes>1Present address: Well-Aging Research Center, Samsung Advanced Institute of Technology, Samsung Electronics, Yongin, Gyeonggido, Korea. E-mail: l.youngsam@gmail.com.</notes><abstract>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 &gt;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 four-subunit 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.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>24449906</pmid><doi>10.1073/pnas.1324001111</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record>
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ispartof Proceedings of the National Academy of Sciences - PNAS, 2014-02, Vol.111 (8), p.2954-2959
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1091-6490
language eng
recordid cdi_jstor_primary_23770863
source JSTOR Archival Journals and Primary Sources Collection; PubMed Central
subjects Biological Sciences
Catalytic activity
Chromatography, Gel
Chromatography, Ion Exchange
Cisplatin - metabolism
DNA
DNA - biosynthesis
DNA Polymerase III - metabolism
DNA Primers - genetics
DNA Repair - physiology
DNA-Binding Proteins - metabolism
DNA-Directed DNA Polymerase - isolation & purification
DNA-Directed DNA Polymerase - metabolism
Fluorescein
Genetic mutation
HEK293 Cells
Holoenzymes - isolation & purification
Holoenzymes - metabolism
Humans
Lesions
Mad2 Proteins - metabolism
Nucleic acids
Nucleotides
Proteins
Saccharomyces cerevisiae
Yeasts
title Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass
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