14-3-3σ is a cruciform DNA binding protein and associates in vivo with origins of DNA replication
14-3-3σ is a cruciform DNA binding protein and associates in vivo with origins of DNA replication
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DOI：
10.1002/jcb.10294
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发表时间：
2002-01-01
影响因子：
4
通讯作者：
Zannis-Hadjopoulos, M
中科院分区：
文献类型：
--
作者：
Alvarez, D;Novac, O;Zannis-Hadjopoulos, M
人类十字形结合蛋白（CBP）是14-3-3蛋白家族的一员，先前已被证明以结构特异性的方式与十字形DNA结合。海关海关检查发现含有14-3-3异构体-，-，-和。在这里，我们通过Western blot分析显示，从带移聚丙烯酰胺凝胶中洗脱的cbp -十字形DNA复合物也含有存在于HeLa细胞核提取物中的143-3sigma异构体。一种针对14-3-3sigma同工异构体的抗体能够干扰cbp -十字形DNA复合物的形成。同样的抗14-3-3sigma抗体对含有猴源ors8最小复制源的质粒p186体外复制的影响也进行了分析。用该抗体预先孵育总HeLa细胞提取物，可将p186的体外复制降低至约30%的对照水平，而非特异性抗体则没有影响。通过染色质免疫沉淀（ChIP）实验（甲醛交联）、抗14-3-3sigma抗体免疫沉淀和定量PCR检测，发现14-3-3sigma在体内以细胞周期依赖的方式与猴DNA复制ors8和ors12起源相关。在G(1)/S期，14-3-3sigma与复制起点的关联最大。结果表明14-3-3sigma是一种通过十字形DNA结合调控DNA复制的起始结合蛋白。
A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 1 4-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding.