Mechanisms of chromosome damage repair in human cells

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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.