1] Istituto Fondazione Italiana per la Ricerca sul Cancro (FIRC) di Oncologia Molecolare (IFOM), Milan, Italy. [2] Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy.
Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland.
Nat Struct Mol Biol. 2014 Oct;21(10):884-92. doi: 10.1038/nsmb.2888. Epub 2014 Sep 7.
Template switching (TS) mediates damage bypass via a recombination-related mechanism involving PCNA polyubiquitination and polymerase δ-dependent DNA synthesis. Using two-dimensional gel electrophoresis and EM, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps of 150-200 nt, and not DNA ends, initiate TS by strand invasion. This causes reannealing of the parental strands and exposure of the nondamaged newly synthesized chromatid, which serves as a replication template for the other blocked nascent strand. Structures resembling double Holliday junctions, postulated to be central double-strand break-repair intermediates but so far visualized only in meiosis, mediate late stages of TS before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA-damage tolerance in mitotic cells and replication under conditions of genotoxic stress.
模板切换（TS）通过涉及 PCNA 多聚泛素化和聚合酶 δ 依赖性 DNA 合成的重组相关机制介导损伤绕过。在这里，我们使用二维凝胶电泳和 EM，在酿酒酵母的一个特定染色体位点上对出现的 TS 中间体进行了表征，确定了五种主要的中间体家族。通过链入侵，150-200nt 的单链 DNA 缺口而不是 DNA 末端引发 TS。这导致亲本链重新退火，并暴露出未受损的新合成染色单体，该染色单体作为另一个受阻的新生链的复制模板。类似于双链 Holliday 连接体的结构，推测是双链断裂修复中间体的核心，但迄今为止仅在减数分裂中观察到，介导 TS 的后期阶段，然后再加工成半嵌合连接体。我们的结果揭示了在有丝分裂细胞中进行重组介导的 DNA 损伤耐受和在遗传毒性应激下进行复制所涉及的 DNA 转换。
