Department of Biochemistry and Molecular Pharmacology, NYU Grossman School of Medicine, New York, NY 10016, USA.
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA.
Mol Cell. 2024 Apr 18;84(8):1460-1474.e6. doi: 10.1016/j.molcel.2024.03.010.
DNA polymerase θ (Polθ) plays a central role in a DNA double-strand break repair pathway termed theta-mediated end joining (TMEJ). TMEJ functions by pairing short-sequence "microhomologies" (MHs) in single-stranded DNA at each end of a break and subsequently initiating DNA synthesis. It is not known how the Polθ helicase domain (HD) and polymerase domain (PD) operate to bring together MHs and facilitate repair. To resolve these transient processes in real time, we utilized in vitro single-molecule FRET approaches and biochemical analyses. We find that the Polθ-HD mediates the initial capture of two ssDNA strands, bringing them in close proximity. The Polθ-PD binds and stabilizes pre-annealed MHs to form a synaptic complex (SC) and initiate repair synthesis. Individual synthesis reactions show that Polθ is inherently non-processive, accounting for complex mutational patterns during TMEJ. Binding of Polθ-PD to stem-loop-forming sequences can substantially limit synapsis, depending on the available dNTPs and sequence context.
DNA 聚合酶 θ（Polθ）在一种称为θ介导的末端连接（TMEJ）的 DNA 双链断裂修复途径中发挥核心作用。TMEJ 通过在断裂两端的单链 DNA 中配对短序列“微同源性（MHs）”，随后启动 DNA 合成。目前尚不清楚 Polθ 解旋酶结构域（HD）和聚合酶结构域（PD）如何协同作用以聚集 MHs 并促进修复。为了实时解析这些瞬态过程，我们利用了体外单分子 FRET 方法和生化分析。我们发现，Polθ-HD 介导最初捕获两条 ssDNA 链，使它们紧密接近。Polθ-PD 结合并稳定预退火的 MH 以形成突触复合物（SC）并启动修复合成。单个合成反应表明，Polθ 固有地无进展性，这解释了 TMEJ 期间复杂的突变模式。Polθ-PD 与茎环形成序列的结合可以根据可用的 dNTP 和序列上下文极大地限制衔接。
