Role of Pif1 family DNA helicase Rrm3 in regulating DNA synthesis during replication stress

项目摘要 细胞在复制应激期间限制DNA复制的能力对于保持基因组完整性至关重要。 我们最近发现，缺乏Rrm 3解旋酶的酵母细胞在生长过程中不会阻止DNA合成。 复制应力我们发现（1）这种新的Rrm 3功能与其解旋酶活性无关， (2)映射到结合orc 5的N-末端尾部的一个区域， 复合物，和（3）N-末端尾部是必不可少的Rrm 3与起源的存在下， 复制应力，但不是在未扰动的细胞。我们假设ORC通过其N-末端尾募集Rrm 3， 复制前复合物，并且这种缔合是复制期间抑制DNA合成所必需的 应力Rrm 3被认为是利用其解旋酶活性来“清扫”复制体前面的DNA， 复制叉进展。因此，我们推断缺乏Rrm 3的酵母是一个很好的模型 用于揭示细胞对复制叉暂停的反应的系统。事实上，使用定量蛋白质组学， 确定了同源重组因子Rdh 54和Rad 5介导的无错误的途径， 损伤旁路在rrm 3缺陷细胞的染色质部分中上调，而缺乏两者的细胞， 和Rad 5积累DNA双链断裂（DSB）。此外，叉保护复杂， 在缺乏Rad 5的细胞中，聚合酶从染色质中丢失。基于这些发现，我们假设， Rad 5定义了一个主要的DSB预防机制，需要克服失速和可能的崩溃 在rrm 3突变体中的暂停分叉。我们进一步假设Rad 5通过介导PCNA来实现这一点。 多聚泛素化，以调节叉块的无错误旁路，如DNA结合蛋白，积累在 在不存在Rrm 3清除酶活性的情况下，和（ii）通过稳定复制体组分， 需要协调重启。为检验这些假设而设计的实验将（1）确定 Rrm 3在复制应激期间限制DNA合成的机制，（2）通过以下方式确定机制： 其中Rrm 3-Orc 5结合调节复制过程中的起点关联、起点活性和DNA合成， 应激和（3）定义对增加的复制叉暂停的细胞应答。我们希望， 这一建议的目的将使人们对维持DNA完整性的基本机制有新的认识 真核细胞中的复制起始和延伸。我们希望我们的研究结果将RRM 3确定为 它不仅是复制体的组成部分，也是起始前复合物的组成部分。我们从 Rrm 3在防止复制叉块中的作用以及Rad 5和Rdh 54在修复这些复制叉块中的作用 通过一种无错误的机制阻止复制叉也将有助于阐明人类细胞如何处理复制叉 阻断并更好地定义Rad 5直系同源物HLTF在抑制肿瘤发生中的作用。

PROJECT SUMMARY The ability of cells to restrict DNA replication during replication stress is critical to preserving genome integrity. We recently discovered that yeast cells lacking the Rrm3 helicase do not arrest DNA synthesis during replication stress. We found (1) that this new Rrm3 function is independent of its helicase activity and instead (2) maps to a region of the poorly characterized N-terminal tail that binds Orc5 of the origin recognition complex, and (3) that the N-terminal tail is essential for Rrm3 association with origins in the presence of replication stress, but not in unperturbed cells. We hypothesize that ORC recruits Rrm3 via its N-terminal tail to pre-replication complexes and that this association is required for inhibition of DNA synthesis during replication stress. Rrm3 is thought to use its helicase activity to ‘sweep’ the DNA ahead of the replisome clear to aid replication fork progression. We reasoned therefore that yeast that lacks Rrm3 makes an excellent model system for revealing the cellular response to replication fork pausing. Indeed, using quantitative proteomics we determined that the homologous recombination factor Rdh54 and the Rad5-mediated pathway for error-free lesion bypass are upregulated in the chromatin fraction of rrm3-deficient cells and that cells lacking both, Rrm3 and Rad5, accumulate DNA double strand breaks (DSBs). Moreover, the fork protection complex and polymerase are lost from the chromatin in cells lacking Rad5. Based on these findings we hypothesize that Rad5 defines a major DSB prevention mechanism that is required to overcome stalling and possibly collapse of paused forks in the rrm3∆ mutant. We further hypothesize that Rad5 accomplishes this by mediating PCNA polyubiquitination to regulate error-free bypass of fork blocks, such as DNA-bound proteins that accumulate on DNA in the absence of the Rrm3 sweepase activity, and (ii) by stabilizing replisome components that are required for coordinated restart. The experiments designed to test these hypotheses will (1) identify the mechanism by which Rrm3 restricts DNA synthesis during replication stress, (2) determine the mechanism by which Rrm3-Orc5 binding regulates origin association, origin activity, and DNA synthesis during replication stress and (3) define the cellular response to increased replication fork pausing. We expect that accomplishing the aims of this proposal will shed new light on fundamental mechanisms that maintain the integrity of DNA replication initiation and elongation in eukaryotic cells. We expect our findings to establish Rrm3 as a component not only of the replisome, but also of the pre-initiation complex at origins. What we learn about the role of Rrm3 in preventing replication fork blocks and about the role of Rad5 and Rdh54 in repairing these blocked forks by an error-free mechanism will also help to clarify how human cells deal with replication fork blocks and better define the role of the Rad5 ortholog HLTF in suppressing tumorigenesis.