Department of Biotechnology, Indian Institute of Technology Kharagpur, India.
International Institute of Molecular and Cell Biology in Warsaw, Poland.
Nucleic Acids Res. 2021 Nov 18;49(20):11834-11854. doi: 10.1093/nar/gkab968.
RECQ1 is the shortest among the five human RecQ helicases comprising of two RecA like domains, a zinc-binding domain and a RecQ C-terminal domain containing the winged-helix (WH). Mutations or deletions on the tip of a β-hairpin located in the WH domain are known to abolish the unwinding activity. Interestingly, the same mutations on the β-hairpin of annealing incompetent RECQ1 mutant (RECQ1T1) have been reported to restore its annealing activity. In an attempt to unravel the strand annealing mechanism, we have crystallized a fragment of RECQ1 encompassing D2-Zn-WH domains harbouring mutations on the β-hairpin. From our crystal structure data and interface analysis, we have demonstrated that an α-helix located in zinc-binding domain potentially interacts with residues of WH domain, which plays a significant role in strand annealing activity. We have shown that deletion of the α-helix or mutation of specific residues on it restores strand annealing activity of annealing deficient constructs of RECQ1. Our results also demonstrate that mutations on the α-helix induce conformational changes and affects DNA stimulated ATP hydrolysis and unwinding activity of RECQ1. Our study, for the first time, provides insight into the conformational requirements of the WH domain for efficient strand annealing by human RECQ1.
RECQ1 是五种人类 RecQ 解旋酶中最短的一种，由两个 RecA 样结构域、一个锌结合结构域和一个含有翼状螺旋（WH）的 RecQ C 端结构域组成。位于 WH 结构域中β发夹尖端的突变或缺失已知会破坏解旋活性。有趣的是，在退火能力不足的 RECQ1 突变体（RECQ1T1）的β发夹上的相同突变已被报道恢复其退火活性。为了揭示链退火机制，我们对包含 D2-Zn-WH 结构域的 RECQ1 片段进行了结晶，该片段在β发夹上带有突变。从我们的晶体结构数据和界面分析中，我们已经证明锌结合结构域中的一个α螺旋可能与 WH 结构域的残基相互作用，这在链退火活性中起着重要作用。我们已经表明，删除α螺旋或突变其特定残基可以恢复 RECQ1 的退火缺陷构建体的链退火活性。我们的结果还表明，α螺旋上的突变会诱导构象变化，并影响 DNA 刺激的 ATP 水解和 RECQ1 的解旋活性。我们的研究首次提供了有关 WH 结构域对人 RECQ1 有效链退火的构象要求的见解。
