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A DNA helicase purified by replication protein A (RPA) affinity chromatography from mouse FM3A cells.

机译:通过从小鼠FM3A细胞复制蛋白A(RPA)亲和层析纯化的DNA解旋酶。

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摘要

In an effort to identify cellular helicases that mimic the action of SV40 large T-antigen, we performed replication protein A (RPA) affinity chromatography on cell extracts from the mouse mammary carcinoma cell line FM3A. In this way, a novel DNA helicase was isolated and purified to near homogeneity. The most purified fractions showed the presence of two proteins of 28 and 21 kDa. Both proteins interacted with 32P-labeled partially duplex DNA when bound to nitrocellulose membranes and were efficiently UV crosslinked to [alpha-32P]dATP. Helicase activity was strongly stimulated by RPA on DNA substrates containing duplex regions longer than 18 bp. Only weak stimulation was observed in the presence of Escherichia coli single strand DNA binding protein (SSB). The enzyme unwinds DNA in the 5'-3' direction in relation to the strand to which it binds. Only ATP and dATP were efficient as nucleoside triphosphate co-factors, and showed similar Km values of approximately 0.6 mM. The properties of this enzyme suggest that it may take part in reactions mediated by RPA such as those predicted to occur at replication forks or alternatively may function during DNA repair or recombination.
机译:为了确定模仿SV40大T抗原作用的细胞解旋酶,我们对小鼠乳腺癌细胞系FM3A的细胞提取物进行了复制蛋白A(RPA)亲和层析。以这种方式,分离出新的DNA解旋酶并将其纯化至接近同质。最纯化的级分显示​​存在28和21 kDa的两种蛋白质。当结合到硝酸纤维素膜上时,这两种蛋白质都与32P标记的部分双链体DNA相互作用,并有效地与[α-32P] dATP进行紫外线交联。 RPA在含有长于18 bp的双链体区域的DNA底物上强烈刺激了解旋酶活性。在大肠杆菌单链DNA结合蛋白(SSB)的存在下,仅观察到弱刺激。该酶相对于与其结合的链沿5'-3'方向解绕DNA。只有ATP和dATP可以有效地用作三磷酸核苷的辅因子,并显示出约0.6 mM的相似Km值。该酶的性质表明,它可能参与RPA介导的反应,例如预计在复制叉处发生的反应,或者可能在DNA修复或重组期间起作用。

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