Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA.
Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN, USA.
Mol Cell. 2022 Oct 6;82(19):3538-3552.e5. doi: 10.1016/j.molcel.2022.08.007. Epub 2022 Sep 7.
DNA becomes single stranded (ssDNA) during replication, transcription, and repair. Transiently formed ssDNA segments can adopt alternative conformations, including cruciforms, triplexes, and quadruplexes. To determine whether there are stable regions of ssDNA in the human genome, we utilized S1-END-seq to convert ssDNA regions to DNA double-strand breaks, which were then processed for high-throughput sequencing. This approach revealed two predominant non-B DNA structures: cruciform DNA formed by expanded (TA) repeats that accumulate in microsatellite unstable human cancer cell lines and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine mirror repeats common across a variety of cell lines. We show that H-DNA is enriched during replication, that its genomic location is highly conserved, and that H-DNA formed by (GAA) repeats can be disrupted by treatment with a (GAA)-binding polyamide. Finally, we show that triplex-forming repeats are hotspots for mutagenesis. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability.
在复制、转录和修复过程中，DNA 会变成单链（ssDNA）。瞬时形成的 ssDNA 片段可以采用替代构象，包括十字形、三链体和四链体。为了确定人类基因组中是否存在稳定的 ssDNA 区域，我们利用 S1-END-seq 将 ssDNA 区域转化为 DNA 双链断裂，然后进行高通量测序。这种方法揭示了两种主要的非 B 型 DNA 结构：由扩展（TA）重复形成的十字形 DNA，这些重复在微卫星不稳定的人类癌细胞系中积累，以及由同源嘌呤/嘧啶镜像重复形成的 DNA 三链体（H-DNA），这些重复在多种细胞系中都很常见。我们表明，H-DNA 在复制过程中富集，其基因组位置高度保守，并且由（GAA）重复形成的 H-DNA 可以通过用（GAA）结合的聚酰胺处理来破坏。最后，我们表明，三链形成重复是突变的热点。我们的结果鉴定了体内的动态 DNA 二级结构，这些结构导致基因组不稳定性升高。
