Mechanisms of chromosome damage repair in human cells

项目摘要 肿瘤细胞中通过同源重组（HR）进行的DNA修复加速了对肿瘤细胞的耐药性的发展。 化疗和放疗，并导致疾病复发。因此，在HR精通中诱发HR缺陷 肿瘤是一个有前途的策略，以增加DNA靶向治疗的疗效。然而，我们仍然不知道 HR反应的阶段对抑制最敏感，因此最有希望靶向。 然而，在突触和链侵入期间抑制HR途径中间体可能是特别重要的。 有效 我们研究的长期目标是为开发新的HR导向的抗肿瘤药物奠定基础。 癌症治疗学我们项目的中心假设是人类细胞进化出了多种途径 的入侵。这个项目的基本原理是，详细了解的分子机制， 链入侵的多种途径可能提供一个强有力的科学框架， 可以开发癌症治疗。本申请的总体目标是（i）阐明本发明的分子结构。 人细胞中HR中链侵入的多个途径的机制，以及（ii）确定 这些途径中，RAD 51激活剂RAD 51 AP 1、RAD 54 L和RAD 54 B的HR功能相交。 中心假设将通过追求两个具体目标进行检验：1）剖析非上位性和上位性 RAD51 AP 1、RAD54 L和RAD54 B之间的关系;以及2）确定 RAD51AP1-RAD54L和RAD51AP1-RAD54B蛋白复合物。在第一个目标下，同基因人类癌症 细胞系将用于确定RAD 51 AP 1、RAD 54 L和/或RAD 54 B的表型结果 删除。评估功能丧失对细胞毒性的影响的β-内酰胺酶敲除策略和测定， 将使用基因组稳定性、复制和重组。对于第二个目的， 利用无核小体和含核小体的DNA底物进行链侵入， 蛋白复合物形成缺陷的突变体将在细胞存活测定中测试互补作用。 申请人认为，本申请中提出的研究具有创新性，因为它侧重于 揭示了存在于人类中的链入侵的多种途径之间的相互作用， 细胞，RAD51 AP 1和RAD54 L之间的通路内合成相互作用，以及人RAD54 B的作用 拟议的研究是重要的，因为它预计将提供强有力的科学理由， 持续开发靶向链侵入的HR刺激物的抑制剂。在此获得的知识 还具有为开发新型癌症疗法提供新机会的潜力。

PROJECT SUMMARY DNA repair by homologous recombination (HR) in tumor cells accelerates the development of resistance to chemo- and radiotherapy and leads to the recurrence of disease. Hence, inducing HR deficiency in HR-proficient tumors is a promising strategy to increase the efficacy of DNA-targeted therapies. Yet, we still do not know which stage of the HR reaction is the most sensitive to inhibition and consequently the most promising to target. However, inhibition of HR pathway intermediates during synapsis and strand invasion may be particularly effective. The long-term goal of our study is to lay the groundwork for the development of novel HR-directed anti- cancer therapeutics. The central hypothesis of our project is that human cells have evolved multiple pathways of strand invasion. The rationale for this project is that a detailed understanding of the molecular mechanisms of the multiple pathways of strand invasion is likely to offer a strong scientific framework whereby new strategies to cancer therapy can be developed. The overall objectives in this application are to (i) elucidate the molecular mechanisms of the multiple pathways of strand invasion in HR in human cells, and (ii) determine the steps in these pathways in which the HR functions of the RAD51 activators RAD51AP1, RAD54L, and RAD54B intersect. The central hypothesis will be tested by pursuing two specific aims: 1) Dissect the non-epistatic and epistatic relationships between RAD51AP1, RAD54L, and RAD54B; and 2) Determine the functional roles of the RAD51AP1-RAD54L and RAD51AP1-RAD54B protein complexes. Under the first aim, isogenic human cancer cell lines will be used to determine the phenotypic consequences of RAD51AP1, RAD54L and/or RAD54B deletion. Proven knockout strategies and assays to evaluate the effect that loss-of-function has on cytotoxicity, genome stability, replication and recombination will be employed. For the second aim, biochemical assays of strand invasion utilizing nucleosome-free and nucleosome-containing DNA substrates will be carried out, and mutants defective in protein complex formation will be tested for complementation in cell survival assays. The research proposed in this application is innovative in the applicant’s opinion, because it focuses on unraveling the poorly understood interplay between the multiple pathways of strand invasion that exist in human cells, the intra-pathway synthetic interaction between RAD51AP1 and RAD54L, and the role of human RAD54B in HR. The proposed research is significant because it is expected to provide strong scientific justification for the continued development of inhibitors that target HR stimulators of strand invasion. The knowledge gained herein also has the potential of offering new opportunities for the development of novel cancer therapies.