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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism*
  • DNA Replication / physiology*
  • DNA, Archaeal / biosynthesis*
  • DNA, Archaeal / genetics
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism
  • DNA, Superhelical / biosynthesis*
  • DNA, Superhelical / genetics
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Pyrococcus furiosus / genetics
  • Pyrococcus furiosus / metabolism*
  • Replication Origin / physiology*

Substances

  • Archaeal Proteins
  • DNA, Archaeal
  • DNA, Single-Stranded
  • DNA, Superhelical
  • Endonucleases