National Institute on Aging, Baltimore, MD 21224, United States.
DNA Repair (Amst). 2010 Jul 1;9(7):796-804. doi: 10.1016/j.dnarep.2010.04.003. Epub 2010 May 6.
Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer predisposition. Of the three, RecQ4's biological and cellular roles have been least thoroughly characterized. Here we tested the helicase activity of purified human RecQ4 on various substrates. Consistent with recent results, we detected ATP-dependent RecQ4 unwinding of forked duplexes. However, our results provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N-terminal portion, but the helicase domain is solely responsible for the enzyme's unwinding activity. In addition, we demonstrate a novel stimulation of RecQ4's helicase activity by replication protein A, similar to that of RecQ1, BLM, WRN, and RecQ5. Together, these data indicate that specific biochemical activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases.
人类有五个成员的 well conserved RecQ 解旋酶家族：RecQ1、Bloom 综合征蛋白 (BLM)、Werner 综合征蛋白 (WRN)、RecQ4 和 RecQ5，它们都以在维持基因组稳定性方面的作用而闻名。BLM、WRN 和 RecQ4 与过早衰老和癌症易感性有关。在这三个中，RecQ4 的生物学和细胞作用特征描述最少。在这里，我们测试了纯化的人 RecQ4 在各种底物上的解旋酶活性。与最近的结果一致，我们检测到 ATP 依赖性 RecQ4 对分叉双链体的解旋。然而，我们的结果首次提供了证据，表明人 RecQ4 的解旋不依赖于链退火，并且不需要存在过量的 ssDNA。此外，我们证明了 Walker A 基序中保守赖氨酸的点突变会使解旋酶活性丧失，这意味着不是 N 端部分，而是解旋酶结构域仅负责酶的解旋活性。此外，我们证明了复制蛋白 A 对 RecQ4 解旋酶活性的一种新的刺激作用，类似于 RecQ1、BLM、WRN 和 RecQ5。这些数据表明，RecQ4 的特定生化活性和蛋白伴侣与其他 RecQ 解旋酶的生化活性和蛋白伴侣保守。
