Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102-1192, USA.
J Biol Chem. 2012 Apr 6;287(15):11820-32. doi: 10.1074/jbc.M112.341347. Epub 2012 Feb 22.
Several proteins have been shown to catalyze branch migration (BM) of the Holliday junction, a key intermediate in DNA repair and recombination. Here, using joint molecules made by human RAD51 or Escherichia coli RecA, we find that the polarity of the displaced ssDNA strand of the joint molecules defines the polarity of BM of RAD54, BLM, RECQ1, and RuvAB. Our results demonstrate that RAD54, BLM, and RECQ1 promote BM preferentially in the 3'→5' direction, whereas RuvAB drives it in the 5'→3' direction relative to the displaced ssDNA strand. Our data indicate that the helicase activity of BM proteins does not play a role in the heterology bypass. Thus, RAD54 that lacks helicase activity is more efficient in DNA heterology bypass than BLM or REQ1 helicases. Furthermore, we demonstrate that the BLM helicase and BM activities require different protein stoichiometries, indicating that different complexes, monomers and multimers, respectively, are responsible for these two activities. These results define BM as a mechanistically distinct activity of DNA translocating proteins, which may serve an important function in DNA repair and recombination.
已经有几种蛋白质被证明可以催化 Holliday 连接点的分支迁移（BM），Holliday 连接点是 DNA 修复和重组的关键中间产物。在这里，我们使用人源 RAD51 或大肠杆菌 RecA 形成的联合分子，发现联合分子中被取代的单链 DNA 链的极性决定了 RAD54、BLM、RECQ1 和 RuvAB 的 BM 极性。我们的结果表明，RAD54、BLM 和 RECQ1 优先以 3'→5'的方向促进 BM，而 RuvAB 则相对于被取代的单链 DNA 链以 5'→3'的方向驱动 BM。我们的数据表明，BM 蛋白的解旋酶活性在异源跨越中不起作用。因此，缺乏解旋酶活性的 RAD54 在 DNA 异源跨越中比 BLM 或 RECQ1 解旋酶更有效。此外，我们证明 BLM 解旋酶和 BM 活性需要不同的蛋白质化学计量，这表明分别由不同的复合物、单体和多聚体负责这两种活性。这些结果将 BM 定义为一种具有独特机制的 DNA 移位蛋白的活性，它可能在 DNA 修复和重组中发挥重要作用。
