New Mechanisms of Replication Stress Response

复制压力反应的新机制

• 批准号: 8788418
• 负责人: Alessandro Vindigni
• 金额: $ 28.79万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8788418
• 关键词: Address; Affect; Antineoplastic Agents; Biochemical; Biological Assay; Camptothecin; Cell physiology; Cells; DNA; DNA Damage; DNA Topoisomerases; DNA biosynthesis; DNA replication fork; Data; Development; Disease; Electron Microscopy; Exposure to; Fanconi anemia protein; Fiber; Frequencies; Genomic Instability; Genotoxic Stress; Health; Human; Imagery; In Vitro; Knowledge; Light; Link; Literature; Malignant Neoplasms; Mediating; Mitomycins; Modality; Modeling; Molecular; Molecular Target; Mutagens; Normal Cell; Pathway interactions; Pharmaceutical Preparations; Physiological; Play; Process; Proliferating; Proteins; Reaction; Regimen; Repair Complex; Role; Stress; Testing; Topoisomerase; Type I DNA Topoisomerases; base; biological adaptation to stress; cancer cell; cancer therapy; chemotherapeutic agent; chemotherapy; chromatin immunoprecipitation; cytotoxicity; design; dosage; helicase; hydroxyurea; improved; inhibitor/antagonist; innovation; insight; novel; novel therapeutic intervention; nuclease; repaired; response; single molecule

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.

描述（由申请人提供）： 项目概要：本提案的目的是了解基因毒性应激诱导后DNA复制叉逆转和重新启动的机制。分叉反转和重启是一个新兴的模型，用来解释停滞或损坏的复制分叉是如何在复制抑制时被处理的。DNA复制抑制剂广泛用于化疗，但它们对正常细胞也有很高的毒性。因此，了解诱导和进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药物剂量敏感至关重要。抑制DNA拓扑异构酶1（TOP 1）的药物目前用于治疗癌症。最近的研究表明，TOP1抑制剂的细胞毒性是由复制叉逆转调制。我们发现，人RECQ 1解旋酶在TOP1抑制后促进反向叉的重新启动，并且PARP 1的多聚腺苷三磷酸化活性通过限制它们的活性来稳定处于退化状态的叉。 重启RECQ 1。在这个应用程序中，我们表明，复制叉的回归并不限于TOP1抑制。事实上，羟基脲（HU）和丝裂霉素C（MMC）治疗诱导复制叉消退。这些发现表明，分叉逆转和重新启动代表了对不同类型癌症化疗药物的一般反应。因此，阐明管理分叉回归和重启的机制将对我们理解几种化疗方式产生广泛影响。我们的初步研究还表明，依赖于DNA 2核酸酶/解旋酶的DNA加工活性的另外的机制涉及当细胞用HU挑战时的过程。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 诱导我们将使用单分子DNA纤维，电子显微镜和生物化学方法的独特组合来测试这些想法：Aim 1将确定是否通过与TOP1抑制相同的中央PARP 1和RECQ 1依赖性机制发生对HU和MMC治疗的叉逆转和重新启动。目标2将确定反向fork处理如何调解fork重新启动。我们将重点关注人类DNA 2和Mre 11核酸酶，因为它们参与这一过程得到了我们的初步研究结果和现有文献的有力支持。目的3阐明复制叉逆转的机制。 我们假设分叉反转是由选定的因素积极驱动的。我们将确定在此过程中对范科尼贫血蛋白M（FANCM）、HepA相关蛋白（HARP，别名SMARCAL 1）以及人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叉逆转，涉及复制应激反应，并与充分表征的基因组不稳定性疾病有关，但它们在叉逆转和重启中的贡献仍有待确定。这些目标的完成将揭示复制对遗传毒性应激的详细机制。