Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712, USA.
Nucleic Acids Res. 2022 Apr 22;50(7):3911-3921. doi: 10.1093/nar/gkac119.
Homologous recombination-deficient cancers rely on DNA polymerase Theta (Polθ)-Mediated End Joining (TMEJ), an alternative double-strand break repair pathway. Polθ is the only vertebrate polymerase that encodes an N-terminal superfamily 2 (SF2) helicase domain, but the role of this helicase domain in TMEJ remains unclear. Using single-molecule imaging, we demonstrate that Polθ-helicase (Polθ-h) is a highly processive single-stranded DNA (ssDNA) motor protein that can efficiently strip Replication Protein A (RPA) from ssDNA. Polθ-h also has a limited capacity for disassembling RAD51 filaments but is not processive on double-stranded DNA. Polθ-h can bridge two non-complementary DNA strands in trans. PARylation of Polθ-h by PARP-1 resolves these DNA bridges. We conclude that Polθ-h removes RPA and RAD51 filaments and mediates bridging of DNA overhangs to aid in polymerization by the Polθ polymerase domain.
同源重组缺陷型癌症依赖于 DNA 聚合酶 Theta（Polθ）介导的末端连接（TMEJ），这是一种替代的双链断裂修复途径。Polθ 是唯一具有 N 端超家族 2（SF2）解旋酶结构域的脊椎动物聚合酶，但该解旋酶结构域在 TMEJ 中的作用尚不清楚。我们通过单分子成像技术证明，Polθ 解旋酶（Polθ-h）是一种具有高度连续性的单链 DNA（ssDNA）马达蛋白，能够有效地从 ssDNA 上剥离复制蛋白 A（RPA）。Polθ-h 也具有有限的解旋 RAD51 丝的能力，但在双链 DNA 上不具有连续性。Polθ-h 可以在反式中桥接两条非互补的 DNA 链。PARP-1 对 Polθ-h 的 PAR 化可以解决这些 DNA 桥。我们得出结论，Polθ-h 去除 RPA 和 RAD51 丝并介导 DNA 突出端的桥接，以帮助 Polθ 聚合酶结构域进行聚合。
