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. 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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Localized Melting of Duplex DNA by Cdc6/Orc1 at the DNA Replication Origin in the Hyperthermophilic Archaeon Pyrococcus Furiosus

Fujihiko Matsunaga et al. Extremophiles. .

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.

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