Zhang Hengshan, Lawrence Christopher W
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):15954-9. doi: 10.1073/pnas.0504586102. Epub 2005 Oct 24.
Evidence for an error-free DNA damage tolerance process in eukaryotes (also called postreplication repair) has existed for more than two decades, but its underlying mechanism, although known to be different from that in prokaryotes, has remained elusive. We have investigated this mechanism in Saccharomyces cerevisiae, in which it is the major component of the RAD6/RAD18 pathway, by transforming an isogenic set of rad1Delta excision-defective strains with plasmids that carry a single thymine-thymine pyrimidine (6-4) pyrimidinone photoadduct in each strand at staggered positions 28 base pairs apart. C-C mismatches placed opposite each of the T-T photoproducts permit unambiguous detection of the events that can lead to the completion of replication: sister-strand recombination or translesion replication on one or the other strand. Despite the severe block to replication that these lesions impose, we find that more than half of the plasmids were fully replicated in a rad1Delta strain and that >90% of them achieved this end by recombination between partially replicated sister strands within the interlesion region. Approximately 60-70% of these events depended on the error-free component of the RAD6/RAD18 pathway, with the remaining events depended on RAD52; these two processes account for almost all of the recombination, which depended neither on DNA polymerase zeta nor on mismatch repair. We conclude that the error-free component of the RAD6/RAD18 pathway completes replication by a mechanism employing recombination between partially replicated sister strands, possibly by means of transient template strand switching or copy choice.
二十多年来，真核生物中存在无差错DNA损伤耐受过程（也称为复制后修复）的证据一直存在，但其潜在机制虽然已知与原核生物不同，但仍然难以捉摸。我们在酿酒酵母中研究了这一机制，在酿酒酵母中它是RAD6/RAD18途径的主要组成部分，通过用携带单个胸腺嘧啶-胸腺嘧啶嘧啶（6-4）嘧啶酮光加合物的质粒转化一组同基因的rad1Δ切除缺陷菌株，每个光加合物在每条链上错开28个碱基对的位置。与每个T-T光产物相对放置的C-C错配允许明确检测可导致复制完成的事件：一条或另一条链上的姐妹链重组或跨损伤复制。尽管这些损伤对复制造成了严重阻碍，但我们发现超过一半的质粒在rad1Δ菌株中完全复制，其中>90%是通过损伤间区域内部分复制的姐妹链之间的重组实现的。这些事件中约60-70%依赖于RAD6/RAD18途径的无差错成分，其余事件依赖于RAD52；这两个过程几乎占了所有的重组，既不依赖于DNA聚合酶ζ也不依赖于错配修复。我们得出结论，RAD6/RAD18途径的无差错成分通过一种机制完成复制，该机制利用部分复制的姐妹链之间的重组，可能是通过瞬时模板链切换或复制选择。
