The Central Region of BRCA1 Binds Preferentially to Supercoiled DNA
The Central Region of BRCA1 Binds Preferentially to Supercoiled DNA
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DOI：
10.1080/07391102.2009.10507299
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发表时间：
2009-08-01
影响因子：
4.4
通讯作者：
Arrowsmith, Cheryl H.
中科院分区：
文献类型：
--
作者：
Brazda, Vaclav;Jagelska, Eva B.;Arrowsmith, Cheryl H.
BRCA1是一种多功能肿瘤抑制蛋白，与细胞周期、转录、DNA修复和染色质重塑等过程的调节有关。BRCA1的功能可能涉及与大量蛋白质和DNA的相互作用，到目前为止，关于BRCA1与DNA结合的证据只有零散的证据。在这项研究中，我们详细分析了与线性和超卷曲（sc） dna结合的各种BRCA1蛋白结构。我们证明，人类BRCA1的中心区域以天然超螺旋密度与负sc质粒DNA强烈结合，琼脂糖凝胶中sc DNA的电泳阻滞证明了这一点。在相对较低的BRCA1:DNA比率下，BRCA1与sc DNA的结合会导致凝胶上出现一个或多个延迟的DNA带。去除BRCA1后，sc DNA恢复了原有的迁移能力。较高浓度的BRCA1蛋白也以线性状态与相同的DNA结合，导致形成一个模糊的迟滞带。我们的实验不仅表明BRCA1倾向于与sc DNA结合，而且还表明中心区域可能包含至少两个对sc DNA具有强亲和力的有效DNA结合域。讨论了BRCA1新DNA结合活性的生物学意义。
BRCA1 is a multifunctional tumor suppressor protein with implications in regulating processes such as cell cycle, transcription, DNA repair, and chromatin remodeling. The function of BRCA1 likely involves interactions with a vast number of proteins and likewise DNA, To this date there is only fragmentary evidence about BRCA1 binding to DNA. In this study, we provide detailed analyses of various BRCA1 protein constructs binding to linear and Supercoiled (sc) DNAs. We demonstrate that the central region of human BRCA1 binds strongly to negatively sc plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of sc DNA in agarose gels. At relatively low BRCA1:DNA ratios, binding of BRCA1 to sc DNA results in the appearance of one or more retarded DNA bands on the gels. After removal of BRCA1, the original mobility of the sc DNA is recovered. BRCA1 proteins at higher concentrations also bind to the same DNA but in linear state, leading to formation of a smeared retarded band. Our experiments not only demonstrate a preference for BRCA1 binding to sc DNA, but also show that the central region may contain at least two efficient DNA binding domains with strong affinity for sc DNA. The biological implications of the novel DNA binding activities of BRCA1 are discussed.