Department of Microbiology and Molecular Genetics, University of Vermont, 89 Beaumont Ave., Burlington, VT 05405, USA; Department of Therapeutic Radiology, Yale University, New Haven, CT 06510, USA.
Department of Therapeutic Radiology, Yale University, New Haven, CT 06510, USA.
Mol Cell. 2021 Apr 1;81(7):1534-1547.e4. doi: 10.1016/j.molcel.2021.01.021. Epub 2021 Feb 11.
Cancers with hereditary defects in homologous recombination rely on DNA polymerase θ (pol θ) for repair of DNA double-strand breaks. During end joining, pol θ aligns microhomology tracts internal to 5'-resected broken ends. An unidentified nuclease trims the 3' ends before synthesis can occur. Here we report that a nuclease activity, which differs from the proofreading activity often associated with DNA polymerases, is intrinsic to the polymerase domain of pol θ. Like the DNA synthesis activity, the nuclease activity requires conserved metal-binding residues, metal ions, and dNTPs and is inhibited by ddNTPs or chain-terminated DNA. Our data indicate that pol θ repurposes metal ions in the polymerase active site for endonucleolytic cleavage and that the polymerase-active and end-trimming conformations of the enzyme are distinct. We reveal a nimble strategy of substrate processing that allows pol θ to trim or extend DNA depending on the DNA repair context.
具有同源重组遗传缺陷的癌症依赖于 DNA 聚合酶θ（pol θ）来修复 DNA 双链断裂。在末端连接过程中，pol θ 将 5'-切除的断裂末端内部的微同源序列排列整齐。在合成发生之前，一种未知的核酸内切酶会修剪 3'末端。在这里，我们报告说，一种与通常与 DNA 聚合酶相关的校对活性不同的核酸内切酶活性是 pol θ 聚合酶结构域固有的。与 DNA 合成活性一样，核酸内切酶活性需要保守的金属结合残基、金属离子和 dNTPs，并且受到 ddNTP 或链终止 DNA 的抑制。我们的数据表明，pol θ 将金属离子重新用于聚合酶活性位点的内切核酸酶切割，并且酶的聚合酶活性和末端修剪构象是不同的。我们揭示了一种灵活的底物加工策略，该策略允许 pol θ 根据 DNA 修复情况修剪或延伸 DNA。
