Friedrich-Loeffler-Institut, Institute of Animal Welfare and Animal Husbandry, Dornbergstrasse 25-27, Celle, Germany .
DNA Cell Biol. 2012 Feb;31(2):180-6. doi: 10.1089/dna.2011.1278. Epub 2011 Aug 31.
Most known DNA replication origins in vertebrate genomes have been found to occur close to transcriptional promoters. The origin of bidirectional DNA replication of the chicken lysozyme GAS41 locus was identified in a CpG island covering the GAS41 gene promoter. In this study, we generated an α-Orc2 antibody from rabbits immunized with the C-terminal half of Orc2 for studying in vivo Orc2 binding to the lysozyme-GAS41 origin. Using the chromatin immunoprecipitation technique and quantitative real-time polymerase chain reaction, we were able to map the Orc2-binding site to a region of the lysozyme GAS41 origin that contains multiple Sp1/Sp3-binding sites co-mapping with two DNase I hypersensitive sites. Further, knockdown of endogenous Sp1 by RNA interference reduced specific Orc2 binding to the lysozyme GAS41 origin. These results suggest that Sp1 participates in recruiting Orc2 to the origin.
大多数已知的脊椎动物基因组中的 DNA 复制起点都被发现在转录启动子附近。鸡溶菌酶 GAS41 基因双向 DNA 复制的起点是在一个包含 GAS41 基因启动子的 CpG 岛上确定的。在这项研究中，我们用 C 端的半 Orc2 免疫兔子产生了一种 α-Orc2 抗体，用于研究体内 Orc2 与溶菌酶-GAS41 起点的结合。使用染色质免疫沉淀技术和定量实时聚合酶链反应，我们能够将 Orc2 结合位点映射到溶菌酶 GAS41 起点的一个区域，该区域包含多个 Sp1/Sp3 结合位点，与两个 DNA 酶 I 超敏位点共定位。此外，RNA 干扰内源性 Sp1 的敲低降低了溶菌酶 GAS41 起点处特定的 Orc2 结合。这些结果表明 Sp1 参与了将 Orc2 招募到起点。
