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首页> 外文期刊>Journal of cellular biochemistry. >14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication.
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14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication.

机译：14-3-3sigma是一种十字形DNA结合蛋白，在体内与DNA复制起点相关。

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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, varepsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma 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. J. Cell. Biochem. 87: 194-207, 2002. Copyright 2002 Wiley-Liss, Inc.
机译：先前显示人类十字形结合蛋白（CBP）以结构特异性方式结合十字形DNA，并且是14-3-3蛋白家族的成员。已经发现CBP包含14-3-3同工型β，γ，伐普西隆和zeta。在这里，我们通过蛋白质印迹分析表明，从带移聚丙烯酰胺凝胶中洗脱的CBP-十字形DNA复合物还包含14-3-3sigma亚型，该亚型存在于HeLa细胞核提取物中。 14-3-3σ同工型特异的抗体能够干扰CBP-十字形DNA复合物的形成。还分析了相同的抗14-3-3sigma抗体在p186体外复制中的作用，p186是一种包含猴源ors8的最小复制源的质粒。用该抗体预孵育总HeLa细胞提取物，可将p186体外复制降低至对照水平的约30％，而非特异性抗体则无作用。通过染色质免疫沉淀（ChIP）测定法（涉及甲醛交联，然后通过免疫沉淀法），发现14-3-3sigma以体内细胞周期依赖的方式与DNA复制ors8和ors12的猴子起源相关联。抗14-3-3sigma抗体和定量PCR。在G（1）/ S阶段，14-3-3sigma与复制起点的关联最大。结果表明14-3-3sigma是一种起源结合蛋白，通过十字形DNA结合参与DNA复制的调控。 J.细胞。生化。 87：194-207，2002。版权所有2002 Wiley-Liss，Inc.。

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