DNA double-strand breaks (DSBs) can arise during normal cell metabolism as a consequence of interaction with reactive oxygen species, collapse of stalled replication forks, telomere dysfunction, chromosome breakage during anaphase, or following programmed genomic rearrangements during immune and germ cell maturation. Additionally, DSBs are formed after exposure to cancer therapies such as radiation therapy or chemotherapeutic agents. Cells have evolved pathways, collectively termed the DNA damage response (DDR), to sense, signal, and repair these lesions. Failure to repair DSBs properly is associated with cancer development, radiation sensitivity, immune deficiencies, and developmental disabilities. We hypothesize that the proteins recruited at DSBs - the DSB proteome - dictate the DNA damage response, including the modality of DSB repair. In particular, we propose that the initial recruitment of the MRN or KU complexes at DSBs influences the quantitative and qualitative landscape of proteins that assemble subsequently in DNA damage and repair foci, as well as their kinetics of assembly. To test these hypotheses we will perform 4 aims. In the first aim, we will use highly sensitive quantitative mass spectrometry approaches to document the stoichiometry and kinetics of recruitment of all proteins present at DSBs generated by endonucleases or at DSBs harboring a DNA topoisomerase adduct. In the second aim, we will monitor competition between MRN and KU at DSBs by evaluating the consequences of depleting KU or MRN on the assembly of the DSB proteome. In the third aim, we will monitor the impact of the PIKKs ATM, ATR and DNA-PKcs on the assembly of the DSB proteome. Finally, in the fourth aim, we will validate and initiate the characterization of novel components of the DSB proteome identified in our studies.
The stepwise assembly of proteins at DSBs into DNA damage foci regulates checkpoint signaling and the modality of DSB repair, which influence the outcome of cancer therapies mediated by the generation of DNA damage: radiation therapy and several classes of chemotherapies. Moreover, chromosome rearrangements, which are implicated in the development of cancer, often arise from pathological forms of repair. The goal of this project is to develop a comprehensive understanding of the DSB proteome and of its regulation and shed light on the mechanisms of misrepair- associated chromosome rearrangements and the mode of action of cancer therapies.
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