New Mechanisms of Replication Stress Response

描述(由申请人提供)： 项目摘要：本提案的目的是了解DNA复制分叉逆转和在遗传毒性应激诱导后重新启动的机制。分叉反转和重新启动是一个新兴的模型，用于解释复制抑制时如何处理停滞或损坏的复制分叉。DNA复制抑制剂被广泛用于化疗，但它们对正常细胞也有很高的毒性。因此，了解诱导和执行分叉逆转和重新启动的机制对于确定新的分子靶点以使癌细胞对对正常细胞无毒的较低剂量的药物敏感至关重要。抑制DNA拓扑异构酶1(TOP1)的药物目前被用于治疗癌症。最近的研究表明，TOP1抑制剂的细胞毒性是通过复制叉逆转来调节的。我们发现，人的RECQ1解旋酶促进TOP1抑制后反向叉子的重新启动，而PARP1的聚(ADPriBosyl)活性通过限制其退化状态稳定叉子。 通过RECQ1重新启动。在本申请中，我们表明复制分叉的回归并不局限于TOP1抑制。的确，羟基脲(HU)和丝裂霉素C(MMC)治疗都会导致复制分叉倒退。这些发现表明，分叉反转和重新启动代表了对不同类别癌症化疗药物的普遍反应。因此，阐明控制分叉倒退和重新启动的机制将对我们理解几种化疗方式产生广泛的影响。我们的初步研究还显示，当细胞受到HU挑战时，依赖于DNA2核酸酶/解旋酶的DNA处理活性的另一种机制参与了这一过程。因此，我们假设至少有两种替代机制控制复制应激后反向分叉的重新启动 归纳法。我们将使用单分子DNA纤维、电子显微镜和生化方法的独特组合来测试这些想法：Aim 1将确定响应HU和MMC处理的分叉反转和重新启动是否通过与响应TOP1抑制时相同的中心PARP1和RECQ1依赖的机制发生。目标2将确定反向分叉处理如何调解分叉重新启动。我们将重点关注人类DNA2和Mre11核酸酶，因为我们的初步发现和现有文献强烈支持它们参与这一过程。目的3将阐明复制分叉逆转的机制。 我们假设，分叉反转是由选定的因素积极推动的。我们将确定在这一过程中对Fanconi贫血蛋白M(FANCM)、HEPA相关蛋白(HARP，别名SMARCAL1)以及人类BLM和WRN解旋酶的需求。这些解旋酶是强有力的候选者，因为它们在体外促进复制分叉逆转，与复制应激反应有关，并与充分描述的基因组不稳定疾病有关，但它们在分叉逆转和重新启动中的作用仍有待确定。这些目标的完成将为复制反应遗传毒性应激的详细机制提供新的线索。

DESCRIPTION (provided by applicant): Project Summary: The objective of this proposal is to understand the mechanisms that govern DNA replication fork reversal and restart following genotoxic stress induction. Fork reversal and restart is an emerging model to explain how stalled or damaged replication forks are processed upon replication inhibition. DNA replication inhibitors are widely used for chemotherapy, but they are also highly toxic to normal cells. Understanding the mechanisms that induce and carry out fork reversal and restart is thus critical for identifying novel molecular targets to sensitize caner cells to lower drug dosages that are not toxic to normal cells. Drugs that inhibit DNA topoisomerase 1 (TOP1) are currently used to treat cancer. Recent studies revealed that TOP1 inhibitor cytotoxicity is modulated by replication fork reversal. We discovered that the human RECQ1 helicase promotes the restart of reversed forks after TOP1 inhibition, and that the poly(ADPribosyl)ation activity of PARP1 stabilizes forks in their regressed state by limiting their restart by RECQ1. Within this application we show that regression of replication forks is not limited to TOP1 inhibition. Indeed, both hydroxyurea (HU) and mitomycin C (MMC) treatment induce replication fork regression. These findings suggest that fork reversal and restart represents a general response to different classes of cancer chemotherapeutics. Thus, elucidating the mechanisms that govern fork regression and restart will have broad impact in our understanding of several chemotherapeutic modalities. Our initial studies also revealed that an additional mechanism relying on the DNA processing activity of the Dna2 nuclease/helicase is implicated in the process when cells are challenged with HU. Therefore, we hypothesize that at least two alternative mechanisms control the restart of reversed forks following replication stress induction. We will test these ideas using a unique combination of single-molecule DNA fiber, electron microscopy, and biochemical approaches: Aim 1 will determine whether fork reversal and restart in response to HU and MMC treatment occurs via the same central PARP1- and RECQ1-dependent mechanism seen in response to TOP1 inhibition. Aim 2 will determine how reversed fork processing mediates fork restart. We will focus on the human Dna2 and Mre11 nucleases since their involvement in this process is strongly supported by our preliminary findings and available literature. Aim 3 will elucidate the mechanism of replication fork reversal. We hypothesize that fork reversal is actively driven by selected factors. We will determine the requirement for Fanconi anemia protein M (FANCM), HepA-related protein (HARP, alias SMARCAL1), as well as the human BLM and WRN helicases, in this process. These helicases are strong candidates because they promote replication fork reversal in vitro, are implicated in replication stress response, and are linked to well-characterized genome-instability disorders, but their contribution in fork reversal and restart remains to be determined. Completion of these aims will shed new light on the detailed mechanisms by which replication responds to genotoxic stress.