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 肿瘤是提高DNA靶向治疗功效的一种有前途的策略。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 RAD 51激活剂RAD 51 AP 1、RAD 54 L和RAD 54 B的HR功能在这些途径中交叉。 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 RAD 51 AP 1、RAD 54 L和RAD 54 B之间的关系;以及2）确定 RAD 51 AP 1-RAD 54 L和RAD 51 AP 1-RAD 54 B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。 本行政补充的目的是获得更换有缺陷和 在我们的生物化学实验中，为了达到所需的蛋白质纯度， 在原裁决的目标2范围内进行调查。

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 objective of this Administrative Supplement is to obtain the possibility to replace defective and outdated equipment necessary to achieve the desired purity of the proteins tested in our biochemical investigations within Aim 2 of the parent award.