PDF(Pubmed)
Abstract：
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.
摘要：
DNA聚合酶θ（Polθ）在称为theta介导的末端连接（TMEJ）的DNA双链断裂修复途径中起着核心作用。TMEJ通过在断裂的每个末端配对单链DNA中的短序列“微同源”（MHs）并随后启动DNA合成来发挥功能。尚不清楚Pol9解旋酶结构域(HD)和聚合酶结构域(PD)如何操作以将MHs结合在一起并促进修复。为了实时解决这些瞬态过程，我们利用体外单分子FRET方法和生化分析.我们发现Polθ-HD介导了两条ssDNA链的初始捕获，让他们靠近。Pol0-PD结合并稳定预退火的MHs以形成突触复合物(SC)并启动修复合成。个别合成反应表明，Pole本质上是非进行性的，解释TMEJ期间复杂的突变模式。Pole-PD与茎环形成序列的结合可以基本上限制突触，取决于可用的dNTP和序列上下文。
