Carvajal-Maldonado Denisse, Li Yuzhen, Returan Mark, Averill April M, Doublié Sylvie, Wood Richard D
Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Center, Houston, Texas, USA.
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA.
J Biol Chem. 2024 Jul;300(7):107461. doi: 10.1016/j.jbc.2024.107461. Epub 2024 Jun 12.
Theta-mediated end joining (TMEJ) is critical for survival of cancer cells when other DNA double-stranded break repair pathways are impaired. Human DNA polymerase theta (Pol θ) can extend ssDNA oligonucleotides, but little is known about preferred substrates and mechanism. We show that Pol θ can extend both ssDNA and RNA substrates by unimolecular stem-loop synthesis initiated by only two 3' terminal base pairs. Given sufficient time, Pol θ uses alternative pairing configurations that greatly expand the repertoire of sequence outcomes. Further primer-template adjustments yield low-fidelity outcomes when the nucleotide pool is imbalanced. Unimolecular stem-loop synthesis competes with bimolecular end joining, even when a longer terminal microhomology for end joining is available. Both reactions are partially suppressed by the ssDNA-binding protein replication protein A. Protein-primer grasp residues that are specific to Pol θ are needed for rapid stem-loop synthesis. The ability to perform stem-loop synthesis from a minimally paired primer is rare among human DNA polymerases, but we show that human DNA polymerases Pol η and Pol λ can catalyze related reactions. Using purified human Pol θ, we reconstituted in vitro TMEJ incorporating an insertion arising from a stem-loop extension. These activities may help explain TMEJ repair events that include inverted repeat sequences.
当其他DNA双链断裂修复途径受损时，θ介导的末端连接（TMEJ）对癌细胞的存活至关重要。人类DNA聚合酶θ（Pol θ）可以延伸单链DNA寡核苷酸，但对其偏好的底物和机制了解甚少。我们发现，Pol θ可以通过仅由两个3'末端碱基对起始的单分子茎环合成来延伸单链DNA和RNA底物。在足够的时间下，Pol θ会使用替代配对构型，这极大地扩展了序列结果的范围。当核苷酸库不平衡时，进一步的引物-模板调整会产生低保真度的结果。单分子茎环合成与双分子末端连接相互竞争，即使存在更长的用于末端连接的末端微同源性也是如此。这两种反应都被单链DNA结合蛋白复制蛋白A部分抑制。快速茎环合成需要Pol θ特有的蛋白质-引物抓握残基。从最小配对引物进行茎环合成的能力在人类DNA聚合酶中很少见，但我们发现人类DNA聚合酶Pol η和Pol λ可以催化相关反应。使用纯化的人类Pol θ，我们在体外重建了TMEJ，其中包含由茎环延伸产生的插入。这些活性可能有助于解释包括反向重复序列的TMEJ修复事件。
