Mechanisms of chromosome damage repair in human cells

项目总结 肿瘤细胞DNA同源重组(HR)修复加速抗药性的形成 化疗和放射治疗会导致疾病复发。因此，导致人力资源熟练者的人力资源不足 肿瘤是提高DNA靶向治疗效果的一种很有前途的策略。然而，我们仍然不知道是哪一个 HR反应阶段对抑制最敏感，因此最有希望成为靶点。 然而，在突触和链侵袭过程中，HR途径中间产物的抑制可能是特别的 有效。 我们研究的长期目标是为开发新型的HR定向反义基因奠定基础 癌症治疗学。我们项目的中心假设是人类细胞进化了多种途径 海滨入侵的证据。这个项目的基本原理是，对分子机制的详细了解 链侵袭的多种途径可能提供一个强有力的科学框架，从而使新的战略 癌症疗法是可以开发的。本申请的总体目标是(I)阐明分子 人类细胞HR中链侵袭的多条途径的机制，以及(Ii)确定 RAD51激活子RAD51AP1、RAD54L和RAD54B的HR功能在这些途径中相交。 核心假设将通过追求两个具体目标来检验：1)剖析非上位性和上位性 RAD51AP1、RAD54L和RAD54B之间的关系；和2)确定RAD51AP1、RAD54L和RAD54B的功能角色 RAD51AP1-RAD54L和RAD51AP1-RAD54B蛋白复合体。在第一个目标下，同基因人类癌症 将使用细胞系来确定RAD51AP1、RAD54L和/或RAD54B的表型结果 删除。经过验证的基因敲除策略和评估功能丧失对细胞毒性的影响的分析方法， 基因组的稳定性、复制和重组将被采用。对于第二个目标，生化分析 将利用无核小体和含有核小体的DNA底物进行链入侵，以及 在蛋白质复合体形成方面有缺陷的突变将在细胞存活分析中进行互补测试。 在申请者看来，本申请中提出的研究是创新的，因为它侧重于 解开人类中存在的多条链侵袭途径之间鲜为人知的相互作用 细胞、RAD51AP1和RAD54L的途径内合成相互作用以及人RAD54B的作用 在人力资源部门。这项拟议的研究意义重大，因为它有望为 继续开发针对链侵袭的HR刺激物的抑制剂。在此获得的知识 也有可能为开发新的癌症疗法提供新的机会。

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.