Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland.
Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland.
Life Sci Alliance. 2020 Feb 18;3(3). doi: 10.26508/lsa.201900547. Print 2020 Mar.
The iron-sulfur (FeS) cluster helicase DDX11 is associated with a human disorder termed Warsaw Breakage Syndrome. Interestingly, one disease-associated mutation affects the highly conserved arginine-263 in the FeS cluster-binding motif. Here, we demonstrate that the FeS cluster in DDX11 is required for DNA binding, ATP hydrolysis, and DNA helicase activity, and that arginine-263 affects FeS cluster binding, most likely because of its positive charge. We further show that DDX11 interacts with the replication factors DNA polymerase delta and WDHD1. In vitro, DDX11 can remove DNA obstacles ahead of Pol δ in an ATPase- and FeS domain-dependent manner, and hence generate single-stranded DNA. Accordingly, depletion of DDX11 causes reduced levels of single-stranded DNA, a reduction of chromatin-bound replication protein A, and impaired CHK1 phosphorylation at serine-345. Taken together, we propose that DDX11 plays a role in dismantling secondary structures during DNA replication, thereby promoting CHK1 activation.
铁硫簇解旋酶 DDX11 与一种被称为华沙破碎综合征的人类疾病有关。有趣的是，一种与疾病相关的突变影响了铁硫簇结合基序中高度保守的精氨酸-263。在这里，我们证明了 DDX11 中的铁硫簇对于 DNA 结合、ATP 水解和 DNA 解旋酶活性是必需的，并且精氨酸-263 影响铁硫簇结合，很可能是因为其正电荷。我们进一步表明，DDX11 与复制因子 DNA 聚合酶 delta 和 WDHD1 相互作用。在体外，DDX11 可以以 ATP 酶和铁硫域依赖性的方式在 Pol δ 之前去除 DNA 障碍物，并因此产生单链 DNA。因此，DDX11 的耗竭会导致单链 DNA 水平降低、染色质结合的复制蛋白 A 减少以及 CHK1 在丝氨酸-345 处的磷酸化受损。总之，我们提出 DDX11 在 DNA 复制过程中发挥作用，以拆除二级结构，从而促进 CHK1 的激活。
