Replication protein A physically interacts with the Bloom's syndrome protein and stimulates its helicase activity
Replication protein A physically interacts with the Bloom's syndrome protein and stimulates its helicase activity
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DOI：
10.1074/jbc.m001557200
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发表时间：
2000-08-04
影响因子：
4.8
通讯作者：
Bohr, VA
中科院分区：
文献类型：
--
作者：
Brosh, RM;Li, JL;Bohr, VA
布卢姆综合征是一种罕见的常染色体隐性遗传疾病，其特征是基因组不稳定和易患癌症。Bloom综合征中的缺陷基因BLM编码一个159-kDa的蛋白，具有DNA刺激的ATP酶和ATP依赖的DNA解旋酶活性。通过分析不同长度的DNA辅因子对ATP酶活性的影响，我们证明BLM是以沿着单链DNA进行性转位的。BLM蛋白催化的解旋酶反应被检查为双链体DNA长度的函数。我们表明，BLM：催化短DNA双链体（小于或等于71个碱基对（bp））的解旋，但严重损害较长DNA双链体（大于或等于259-bp）。人单链DNA结合蛋白（人复制蛋白A（hRPA））的存在刺激259-bp部分双链体DNA底物上的BLM解旋反应。异源单链DNA结合蛋白不能类似地刺激BLM蛋白的解旋酶活性。这是BLM和另一种蛋白质之间的功能性相互作用的第一个证明。与hRPA和BLM解旋酶之间的功能性相互作用一致，我们证明了RPA的70 kDa亚基介导的两种蛋白质之间的直接物理相互作用。BLM和hRPA之间的相互作用表明，这两种蛋白质在体内一起发挥作用，在复制，重组或修复过程中解开DNA双链体。
Bloom's syndrome is a rare autosomal recessive disorder characterized by genomic instability and predisposition to cancer. BLM, the gene defective in Bloom's syndrome, encodes a 159-kDa protein possessing DNA-stimulated ATPase and ATP-dependent DNA helicase activities. We have examined mechanistic aspects of the catalytic functions of purified recombinant BLM protein, Through analyzing the effects of different lengths of DNA cofactor on ATPase activity, we provide evidence to suggest that BLM translocates along single-stranded DNA in a processive manner. The helicase reaction catalyzed by BLM protein was examined as a function of duplex DNA length. We show that BLM: catalyzes unwinding of short DNA duplexes (less than or equal to 71 base pairs (bp)) but is severely compromised on longer DNA duplexes (greater than or equal to 259-bp), The presence of the human single-stranded DNA-binding protein (human replication protein A (hRPA)) stimulates the BLM unwinding reaction on the 259-bp partial duplex DNA substrate. Heterologous single-stranded DNA-binding proteins fail to stimulate similarly the helicase activity of BLM protein. This is the first demonstration of a functional interaction between BLM and another protein. Consistent with a functional interaction between hRPA and the BLM helicase, we demonstrate a direct physical interaction between the two proteins mediated by the 70-kDa subunit of RPA. The interactions between BLM and hRPA suggest that the two proteins function together in vivo to unwind DNA duplexes during replication, recombination, or repair.