Institute of Molecular Biology, 55128 Mainz, Germany.
Institute of Molecular Biology, 55128 Mainz, Germany.
Mol Cell. 2020 Jan 2;77(1):3-16.e4. doi: 10.1016/j.molcel.2019.09.015. Epub 2019 Oct 10.
Tracing DNA repair factors by fluorescence microscopy provides valuable information about how DNA damage processing is orchestrated within cells. Most repair pathways involve single-stranded DNA (ssDNA), making replication protein A (RPA) a hallmark of DNA damage and replication stress. RPA foci emerging during S phase in response to tolerable loads of polymerase-blocking lesions are generally thought to indicate stalled replication intermediates. We now report that in budding yeast they predominantly form far away from sites of ongoing replication, and they do not overlap with any of the repair centers associated with collapsed replication forks or double-strand breaks. Instead, they represent sites of postreplicative DNA damage bypass involving translesion synthesis and homologous recombination. We propose that most RPA and recombination foci induced by polymerase-blocking lesions in the replication template are clusters of repair tracts arising from replication centers by polymerase re-priming and subsequent expansion of daughter-strand gaps over the course of S phase.
通过荧光显微镜追踪 DNA 修复因子，可以提供有关细胞内 DNA 损伤处理如何协调的有价值信息。大多数修复途径都涉及单链 DNA（ssDNA），因此复制蛋白 A（RPA）成为 DNA 损伤和复制应激的标志。在 S 期，当细胞受到可容忍的聚合酶阻断损伤时，会出现 RPA 焦点，通常被认为是停滞的复制中间体。我们现在报告说，在芽殖酵母中，它们主要远离正在进行复制的部位形成，并且它们与与崩溃的复制叉或双链断裂相关的任何修复中心都不重叠。相反，它们代表涉及跨损伤合成和同源重组的复制后 DNA 损伤绕过的部位。我们提出，在复制模板中由聚合酶阻断损伤诱导的大多数 RPA 和重组焦点是修复道的簇，这些簇是由复制中心通过聚合酶重新引发和随后在 S 期过程中扩大子链缺口产生的。
