Li Yuzhen, Dang Ngoc K, He Wei, Returan Mark, Carvajal-Maldonado Denisse, Guerin Adele T, Xu Han, Liu Bin, Wood Richard D
Department of Epigenetics and Molecular Carcinogenesis, MD Anderson Cancer Center, Houston, TX, USA.
Nat Commun. 2025 Jul 2;16(1):6085. doi: 10.1038/s41467-025-61258-3.
DNA polymerase theta (Pol θ) initiates repair of DNA double-strand breaks by pairing single strands at short "microhomologies". It is important to understand microhomology selection, as some cancer cells rely on Pol θ for survival. Here, we investigate end-joining by purified human Pol θ, employing DNA sequencing of products generated from oligonucleotide libraries having diverse 3' ends. Pol θ overwhelmingly selects short internal microhomologies found within 15 nucleotides of the terminus of single-stranded DNAs, restricting deletion size during end-joining. Significantly, we find that the selected microhomologies are usually interrupted by mismatches and that base pairing within 6 nucleotides of the 3' end is important for determining microhomology choice. Bidirectional synthesis is not necessary to initiate end-joining. The preference for mismatched microhomologies suggests a revision of the definition of microhomology to account for the unique properties of Pol θ. This could advance the analysis of mutations in cancer genomes.
DNA聚合酶θ（Pol θ）通过在短的“微同源性”处配对单链来启动DNA双链断裂的修复。了解微同源性选择很重要，因为一些癌细胞依靠Pol θ来生存。在这里，我们利用具有不同3'端的寡核苷酸文库产生的产物进行DNA测序，研究纯化的人Pol θ的末端连接。Pol θ绝大多数选择在单链DNA末端15个核苷酸内发现的短内部微同源性，限制了末端连接过程中的缺失大小。值得注意的是，我们发现所选的微同源性通常被错配打断，并且3'端6个核苷酸内的碱基配对对于确定微同源性选择很重要。双向合成对于启动末端连接不是必需的。对错配微同源性的偏好表明需要修订微同源性的定义，以考虑Pol θ的独特特性。这可能会推进对癌症基因组中突变的分析。
