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

Fujihiko Matsunaga; Kie Takemura; Masaki Akita; Akinori Adachi; Takeshi Yamagami; Yoshizumi Ishino

doi:10.1007/s00792-009-0284-9

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

Fujihiko Matsunaga, Kie Takemura, Masaki Akita, Akinori Adachi, Takeshi Yamagami, Yoshizumi Ishino

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

17 Citations (Scopus)

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.

Original language	English
Pages (from-to)	21-31
Number of pages	11
Journal	Extremophiles
Volume	14
Issue number	1
DOIs	https://doi.org/10.1007/s00792-009-0284-9
Publication status	Published - Jan 2010

All Science Journal Classification (ASJC) codes

Microbiology
Molecular Medicine

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title = "Localized melting of duplex DNA by Cdc6/Orc1 at the DNA replication origin in the hyperthermophilic archaeon Pyrococcus furiosus",

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.",

author = "Fujihiko Matsunaga and Kie Takemura and Masaki Akita and Akinori Adachi and Takeshi Yamagami and Yoshizumi Ishino",

note = "Funding Information: Acknowledgments We dedicate this paper to the memory of Yasuo Kawasaki. We thank Dr. Sonoko Ishino for critical reading of the manuscript. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y.I. and F.M.).",

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AU - Matsunaga, Fujihiko

AU - Takemura, Kie

AU - Akita, Masaki

AU - Adachi, Akinori

AU - Yamagami, Takeshi

AU - Ishino, Yoshizumi

N1 - Funding Information: Acknowledgments We dedicate this paper to the memory of Yasuo Kawasaki. We thank Dr. Sonoko Ishino for critical reading of the manuscript. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y.I. and F.M.).

PY - 2010/1

Y1 - 2010/1

N2 - 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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Localized melting of duplex DNA by Cdc6/Orc1 at the DNA replication origin in the hyperthermophilic archaeon Pyrococcus furiosus

Abstract

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