Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Dell Pediatric Research Institute, 1400 Barbara Jordan Blvd. Austin, TX 78723, USA.
Nucleic Acids Res. 2013 Dec;41(22):10345-57. doi: 10.1093/nar/gkt804. Epub 2013 Sep 17.
Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA.
在人类基因组中，具有采用替代（即非 B）DNA 结构能力的序列被认为与刺激基因组不稳定性有关。先前，我们发现天然存在的分子内三链体（H-DNA）在哺乳动物中主要以 DNA 双链断裂的形式引起遗传不稳定性。因此，确定处理 H-DNA 涉及的机制很重要。最近，我们证明了人类 DHX9 解旋酶在体外优先解开分子间三链体 DNA。在此，我们使用含有 H-DNA 的突变报告系统来研究 DHX9 活性在人类细胞中天然存在的 H-DNA 结构中的相关性。我们发现 H-DNA 显著增加了经小干扰 RNA（siRNA）处理、DHX9 耗尽的细胞中的诱变，主要影响缺失。此外，DHX9 与超螺旋质粒细胞中的 H-DNA 相关联。为了进一步研究 DHX9 在 H-DNA 识别/处理中的作用，我们在体外进行了结合测定，并在 U2OS 细胞中进行了染色质免疫沉淀测定。DHX9 识别 H-DNA，证据是它与 H-DNA 结构结合，并与对照区域相比在人类细胞中富集在 H-DNA 区域。这些综合数据表明 DHX9 参与体内 H-DNA 结构的处理，并支持其在替代结构 DNA 位点处维持基因组整体稳定性的作用。
