title: Studies on human DNA polymerase epsilon and GINS complex and their role in DNA replication.
abstract: In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol α, δ, and ε. In this report, we describe the reconstitution of the human four-subunit Pol ε and characterization of its catalytic properties in comparison with Pol α and Pol δ. Human Pol ε holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol ε p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) ε was found more processive than hPol δ in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol ε and hPol δ in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (α, δ, and ε) and to markedly stimulate the activities of Pol α and Pol ε. In vivo studies indicated that siRNA-targeted depletion of hPol δ and/or hPol ε reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol ε had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol δ. We suggest that reduction in the level of Pol δ may be less deleterious because of its collision-and-release role in lagging strand synthesis.
authors.affiliation: Program of Molecular Biology, Memorial Sloan Kettering Cancer Center, New York, New York 10021.
authors.forename: Vladimir P
authors.initials: VP
authors.lastname: Bermudez
authors.affiliation: Program of Molecular Biology, Memorial Sloan Kettering Cancer Center, New York, New York 10021.
authors.forename: Andrea
authors.initials: A
authors.lastname: Farina
authors.affiliation: Program of Molecular Biology, Memorial Sloan Kettering Cancer Center, New York, New York 10021.
authors.forename: Vineetha
authors.initials: V
authors.lastname: Raghavan
authors.affiliation: Program of Molecular Biology, Memorial Sloan Kettering Cancer Center, New York, New York 10021.
authors.forename: Inger
authors.initials: I
authors.lastname: Tappin
authors.affiliation: Program of Molecular Biology, Memorial Sloan Kettering Cancer Center, New York, New York 10021. Electronic address: j-hurwitz@ski.mskcc.org.
authors.forename: Jerard
authors.initials: J
authors.lastname: Hurwitz
mesh_terms: D000818:Animals; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics; D012441:Saccharomyces cerevisiae
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D034741:RNA, Small Interfering; D004247:DNA; D004257:DNA Polymerase II; D004258:DNA Polymerase III