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The DNA polymerase activity of Pol ε holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts

Shikata, Koh and Sasa-Masuda, Taro and Okuno, Yukiko and Waga, Shou and Sugino, Akio (2006) The DNA polymerase activity of Pol ε holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts. BMC Biochemistry, 7 . Art. No. 21. ISSN 1471-2091. PMCID PMC1560149. https://resolver.caltech.edu/CaltechAUTHORS:SHIbmcbiochem06

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[img] Image (JPEG) (Additional file 1: Cloning strategy of Xenopus Pol ε p260 subunit cDNA by 5' RACE) - Supplemental Material
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[img] Image (JPEG) (Additional file 2: Deduced amino acid sequences of Xenopus and human Pol ε p260) - Supplemental Material
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[img] Image (JPEG) (Additional file 3: Structural similarity between Xenopus and S. cerevisiae Pol ε catalytic subunit) - Supplemental Material
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[img] Image (JPEG) (Additional file 4: Cloning of Xenopus Pol ε p17 and p12 subunits) - Supplemental Material
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[img] Image (JPEG) (Additional file 5: xGINS stimulates DNA synthesis catalyzed by xPol ε holoenzyme) - Supplemental Material
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Abstract

Background: DNA polymerase ε (Pol ε) is involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the roles of replicative Pol α and Pol δ in chromosomal DNA replication are relatively well understood and well documented, the precise role of Pol ε in chromosomal DNA replication is not well understood. Results: This study uses a Xenopus egg extract DNA replication system to further elucidate the replicative role(s) played by Pol ε. Previous studies show that the initiation timing and elongation of chromosomal DNA replication are markedly impaired in Pol ε-depleted Xenopus egg extracts, with reduced accumulation of replicative intermediates and products. This study shows that normal replication is restored by addition of Pol ε holoenzyme to Pol ε-depleted extracts, but not by addition of polymerase-deficient forms of Pol ε, including polymerase point or deletion mutants or incomplete enzyme complexes. Evidence is also provided that Pol ε holoenzyme interacts directly with GINS, Cdc45p and Cut5p, each of which plays an important role in initiation of chromosomal DNA replication in eukaryotic cells. Conclusion: These results indicate that the DNA polymerase activity of Pol ε holoenzyme plays an essential role in normal chromosomal DNA replication in Xenopus egg extracts. These are the first biochemical data to show the DNA polymerase activity of Pol ε holoenzyme is essential for chromosomal DNA replication in higher eukaryotes, unlike in yeasts.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://bmcbiochem.biomedcentral.com/articles/10.1186/1471-2091-7-21PublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1560149/PubMed CentralArticle
https://doi.org/10.1186/1471-2091-7-21DOIUNSPECIFIED
https://doi.org/10.1186/1471-2091-7-21DOIUNSPECIFIED
Alternate Title:The DNA polymerase activity of Pol epsilon holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts
Additional Information:© 2006 Shikata et al., licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Submission date 12 May 2006; Acceptance date 22 August 2006; Publication date 22 August 2006. KS carried out the molecular biological and genetic studies, participated in the sequence alignment and drafted the manuscript. TSM carried out the purification of Xenopus proteins from insect cells and immunoprecipitation assays. YO participated in preparation of Xenopus egg extracts. SW participated in the design of the study and discussion. AS conceived of the study, participated in its design and coordination, performed some experiments, and helped to draft the manuscript. All authors read and approved the final manuscript. This work was supported partly by grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan to AS. Five JPEGs included as supplmentary files.
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Ministry of Education, Culture, Sports, Science and Technology (MEXT)UNSPECIFIED
PubMed Central ID:PMC1560149
Record Number:CaltechAUTHORS:SHIbmcbiochem06
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:SHIbmcbiochem06
Official Citation:The DNA polymerase activity of Pol ε holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts Koh Shikata, Taro Sasa-Masuda, Yukiko Okuno, Shou Waga and Akio Sugino BMC Biochemistry 2006 7:21 DOI: 10.1186/1471-2091-7-21
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:4545
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:29 Aug 2006
Last Modified:02 Oct 2019 23:14

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