DNA end processing by the Mre11/Rad50/Nbs1 complex in human cells

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Double-strand breaks in DNA are a lethal form of genome damage that can also lead to large- scale chromosomal rearrangements and deletions when mis-repaired. Non-homologous end joining and homologous recombination are the two major pathways of DNA double-strand break repair in eukaryotes, and the decision between these pathways can have long-lasting consequences for cell fate. Current models of DNA repair suggest that non-homologous and homologous recombination factors compete with each other for end binding, processing, and repair in a manner that favors homologous recombination during the S and G2 phases of the cell cycle, but this competition is still ill-defined despite many years of study. Here we build upon our recent results showing that the Mre11-Rad50-Nbs1 (MRN) complex performs end processing in physiological conditions that depends on the core non-homologous end joining complex, DNA- dependent protein kinase (DNA-PK). Ensemble biochemistry, single-molecule experiments, and quantitation of DNA repair intermediates in human cells shows that MRN-mediated end processing occurs at DNA-PK-bound ends, promoted by the cell cycle-regulated repair factor CtIP. These results suggest that DNA double-strand break repair "choice" is not a competition, but rather a sequential and ordered process from non-homologous to homologous pathways. To validate these results and understand double-strand break recognition and processing at a mechanistic level, we propose to further investigate the characteristics of DNA end processing globally in human cells. We will test our hypotheses by analyzing the regulation of DNA end processing by by Mre11 nuclease activity and CtIP modifications, and will examine the characteristics of DNA end processing as it occurs in non-cycling cells. Lastly, we will investigate the structural biology of the cooperative, multi-subunit end repair complex that MRN forms with DNA-PK on DNA ends, with both recombinant proteins as well as native complexes from human cells. These experiments will further establish novel methods for characterizing DNA repair intermediates and solidify a new paradigm in DNA double-strand break repair that mechanistically links non-homologous end joining with the initiation of homologous recombination.