Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.
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.
Matsunaga
Fujihiko
F
Institut de Génétique et Microbiologie, UMR8621, Bât. 409, Université Paris-Sud, 91405 Orsay Cedex, France. fujihiko@agr.kyushu-u.ac.jp
Glatigny
Annie
A
Mucchielli-Giorgi
Marie-Hélène
MH
Agier
Nicolas
N
Delacroix
Hervé
H
Marisa
Laetitia
L
Durosay
Patrice
P
Ishino
Yoshizumi
Y
Aggerbeck
Lawrence
L
Forterre
Patrick
P
D019843:Archaeal Proteins / Q000378:metabolism*; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020745:Genome, Archaeal; D020411:Oligonucleotide Array Sequence Analysis; D051738:Origin Recognition Complex / Q000378:metabolism*; D043903:Pyrococcus abyssi / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D018741:Replication Origin
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex