Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.
Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.
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CDC6/ORC1和MCM蛋白的DNA结合活性的基因组和生化分析。
DOI：
10.1093/nar/gkm212
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发表时间：
2007
影响因子：
14.9
通讯作者：
Forterre, Patrick
中科院分区：
文献类型：
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作者：
Matsunaga, Fujihiko;Glatigny, Annie;Mucchielli-Giorgi, Marie-Helene;Agier, Nicolas;Delacroix, Herve;Marisa, Laetitia;Durosay, Patrice;Ishino, Yoshizumi;Aggerbeck, Lawrence;Forterre, Patrick
超嗜热古菌Pyrococcus abyssi的DNA复制起点（oriC）含有多个ORB和mini-ORB重复序列，与其他古菌ORB（起点识别框）具有序列相似性。在这里，我们报告的Cdc 6/Orc 1的结合到一个5 kb的区域含有oriC在体内是高度特异性的指数和稳定期，通过染色质免疫沉淀结合杂交的全基因组微阵列（ChIP芯片）。oriC区域实际上是Cdc 6/Orc 1的唯一结合位点，从而在1.8 M bp基因组中区分oriC。我们发现，5 kb的区域包含一个以前未注意到的ORB和迷你ORB重复序列的基因编码的小亚基（dp 1）的DNA聚合酶II（PolD）的集群。ChIP和凝胶阻滞分析进一步揭示Cdc 6/Orc 1特异性结合oriC和dp 1中的ORB簇。组织的ORB集群在DP 1和oriC是保守的进化过程中的顺序Thermococales，这表明在启动DNA复制的作用。我们的ChIP芯片分析还显示，Mcm根据生长阶段改变了与oriC区域的结合特异性，这与其作为许可因子的作用一致。
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.