Nucleic Acids Research Advance Access originally published online on April 22, 2007
Nucleic Acids Research 2007 35(10):3214-3222; doi:10.1093/nar/gkm212
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Nucleic Acids Research, 2007, Vol. 35, No. 10 3214-3222
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.
1Institut de Génétique et Microbiologie, UMR8621, Bât. 409, Université Paris-Sud, 91405 Orsay Cedex, France, 2Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan and 3Gif/Orsay DNA Microarray Platform (GODMAP), Centre de Génétique Moléculaire UPR2167, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, 4Associated with the Université Pierre et Marie Curie-Paris 6, Paris F-75005, France
*To whom correspondence should be addressed. Tel: +33 1 69 157489; Fax: +33 1 69 157808; Email: patrick.forterre{at}igmors.u-psud.fr
Correspondence may also be addressed to Fujihiko Matsunaga. Tel: +81 92 642 4218; Fax: +81 92 642 3051; Email: fujihiko{at}agr.kyushu-u.ac.jp
Received February 16, 2007. Revised March 26, 2007. Accepted March 27, 2007.
The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor.
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