Localized melting of duplex DNA by Cdc6/Orc1 at the DNA replication origin in the hyperthermophilic archaeon Pyrococcus furiosus.
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.
Department of Genetic Resources Technology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Fukuoka 812-8581, Japan.
Fujihiko
F
Matsunaga
Kie
K
Takemura
Masaki
M
Akita
Akinori
A
Adachi
Takeshi
T
Yamagami
Yoshizumi
Y
Ishino
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D019641:DNA, Archaeal / Q000096:biosynthesis* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000096:biosynthesis* / Q000235:genetics; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin / Q000502:physiology*
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004720:Endonucleases