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Mechanisms of chromosome damage repair in human cells

人体细胞染色体损伤修复机制

基本信息

批准号：
10798638
负责人：
Claudia Wiese
金额：
$ 4.74万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10798638
关键词：
ATP phosphohydrolase Acceleration Administrative Supplement Anaphase Award Biochemical Biological Assay Biological Process C-terminal Cancer cell line Cell Survival Cells Chromosome Pairing Chromosome abnormality Chromosomes Complement Complex Cytoprotection DNA DNA Damage DNA Repair Development Disease Equipment Event Exhibits Fiber Filament Genetic Complementation Test Genetic Recombination Genome Stability Goals Human Individual Invaded Investigation Knock-out Knowledge Malignant Neoplasms Maps Mediating Mission Molecular Mutagens Natural Killer Cells Nucleosomes Outcome Parents Pathway interactions Phenotype Proteins Public Health RAD54L gene RNA Radiation therapy Reaction Recurrent disease Reporter Research Resistance Resistance development Resolution Role Route Single-Stranded DNA Telomere Pathway Testing Ultrafine United States National Institutes of Health anti-cancer therapeutic cancer therapy chemotherapeutic agent chemotherapy cytotoxicity ds-DNA genotoxicity homologous recombination loss of function mutant neoplastic cell novel novel therapeutics paralogous gene postsynaptic protein complex repaired response synthetic construct targeted treatment telomere tumor

项目摘要

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.

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。本行政副刊的目的是获得替换有缺陷和陈旧的设备需要达到我们生化测试中所测试的蛋白质的预期纯度在父母裁决的目标2范围内进行调查。