Regression of Replication Forks Stalled by Leading-strand Template Damage II-REGRESSION BY RecA IS INHIBITED BY SSB
Regression of Replication Forks Stalled by Leading-strand Template Damage II-REGRESSION BY RecA IS INHIBITED BY SSB
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DOI：
10.1074/jbc.m114.587907
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发表时间：
2014-10-10
影响因子：
4.8
通讯作者：
Marians, Kenneth J.
中科院分区：
文献类型：
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作者：
Gupta, Sankalp;Yeeles, Joseph T. P.;Marians, Kenneth J.
停滞的复制叉是染色体断裂和破坏基因组稳定性的毒性重组中间体形成的位点。因此，复制叉修复和重新激活是必不可少的过程。在复制叉再激活的许多模型中，有一种模型调用了由链交换蛋白RecA催化的叉回归，作为处理停滞叉的中间体。我们已经研究了复制叉回归活动RecA使用重建的DNA复制系统，其中复制体是停滞的碰撞与前导链模板损伤。我们发现RecA在复制体和SSB存在的情况下无法使停滞的分叉回归。如果复制蛋白从停滞的分叉中被移除，只要冈崎片段被密封，RecA就会催化净回归。RecA产生的霍利迪连接可以通过RuvC切割来检测，尽管这不是稳健的反应。另一方面，在RuvC活性被抑制的条件下，观察到RecA的广泛分支迁移，其中产生由成对的新生前导链和滞后链组成的完全解绕的产物。该分支迁移反应被SSB抑制，这可能是RecA在复制蛋白存在下不能产生产物的原因。有趣的是，我们发现RecA-RuvC反应得到不同程度的支持，这取决于模板损伤;携带环嘧啶二聚体的模板比携带合成脱碱基位点的模板引发更多的RecA-RuvC产物。这种差异可以归因于更高的亲和力RecA结合到DNA携带胸苷二聚体比那些与脱碱基位点。
Stalled replication forks are sites of chromosome breakage and the formation of toxic recombination intermediates that undermine genomic stability. Thus, replication fork repair and reactivation are essential processes. Among the many models of replication fork reactivation is one that invokes fork regression catalyzed by the strand exchange protein RecA as an intermediate in the processing of the stalled fork. We have investigated the replication fork regression activity of RecA using a reconstituted DNA replication system where the replisome is stalled by collision with leading-strand template damage. We find that RecA is unable to regress the stalled fork in the presence of the replisome and SSB. If the replication proteins are removed from the stalled fork, RecA will catalyze net regression as long as the Okazaki fragments are sealed. RecA-generated Holliday junctions can be detected by RuvC cleavage, although this is not a robust reaction. On the other hand, extensive branch migration by RecA, where a completely unwound product consisting of the paired nascent leading and lagging strands is produced, is observed under conditions where RuvC activity is suppressed. This branch migration reaction is inhibited by SSB, possibly accounting for the failure of RecA to generate products in the presence of the replication proteins. Interestingly, we find that the RecA-RuvC reaction is supported to differing extents, depending on the template damage; templates carrying a cyclopyrimidine dimer elicit more RecA-RuvC product than those carrying a synthetic abasic site. This difference could be ascribed to a higher affinity of RecA binding to DNAs carrying a thymidine dimer than to those with an abasic site.