Studies on human DNA polymerase ε and GINS Complex and their role in DNA replication Journal Article
| Authors: | Bermudez, V. P.; Farina, A.; Raghavan, V.; Tappin, I.; Hurwitz, J. |
| Article Title: | Studies on human DNA polymerase ε 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 foursubunit 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 δand/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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | controlled study; protein expression; unclassified drug; gene sequence; human cell; nonhuman; dna directed dna polymerase eta; dna polymerase; dna replication; dna synthesis; protein function; cell proliferation; protein analysis; animals; chromosomal proteins, non-histone; cell cycle progression; protein depletion; protein protein interaction; small interfering rna; rna, small interfering; in vivo study; enzyme activity; hela cells; dna; amino terminal sequence; saccharomyces cerevisiae; eukaryota; nucleotide sequence; in-vivo; helicase; catalysis; dna replications; cycline; protein subunit; protein structure; rna polymerase ii; inhibitory effect; catalytic properties; helicases; catalytic domains; cell-cycle progression; dna virus infection; dna directed dna polymerase alpha; dna directed dna polymerase delta; n-terminals; dna chains; dna synthesiss; eukaryotic cells; human dna polymerase; lagging strand; monomeric complexes; proliferating cells; protein gins; protein p12; protein p17; protein p59; baculovirus; dna polymerase iii; dna polymerase ii |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 286 |
| Issue: | 33 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2011-08-19 |
| Start Page: | 28963 |
| End Page: | 28977 |
| Language: | English |
| DOI: | 10.1074/jbc.M111.256289 |
| PROVIDER: | scopus |
| PMCID: | PMC3190704 |
| PUBMED: | 21705323 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 1" - "Export Date: 3 October 2011" - "CODEN: JBCHA" - "Source: Scopus" |
Altmetric
Citation Impact
BMJ Impact Analytics
Related MSK Work