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Extremophiles. 2010 Jan;14(1):21-31. doi: 10.1007/s00792-009-0284-9. Epub 2009 Sep 30.

Localized melting of duplex DNA by Cdc6/Orc1 at the DNA replication origin in the hyperthermophilic archaeon Pyrococcus furiosus.

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  • 1Department of Genetic Resources Technology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Fukuoka 812-8581, Japan.

Abstract

The initiation step is a key process to regulate the frequency of DNA replication. Although recent studies in Archaea defined the origin of DNA replication (oriC) and the Cdc6/Orc1 homolog as an origin recognition protein, the location and mechanism of duplex opening have remained unclear. We have found that Cdc6/Orc1 binds to oriC and unwinds duplex DNA in the hyperthermophilic archaeon Pyrococcus furiosus, by means of a P1 endonuclease assay. A primer extension analysis further revealed that this localized unwinding occurs in the oriC region at a specific site, which is 12-bp long and rich in adenine and thymine. This site is different from the predicted duplex unwinding element (DUE) that we reported previously. We also discovered that Cdc6/Orc1 induces topological changes in supercoiled oriC DNA, and that this process is dependent on the AAA+ domain. These results indicate that topological alterations of oriC DNA by Cdc6/Orc1 introduce a single-stranded region at the 12-mer site, that could possibly serve as an entry point for Mcm helicase.

PMID:
19787415
DOI:
10.1007/s00792-009-0284-9
[PubMed - indexed for MEDLINE]

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