Tuesday, April 1, 2025
4/1/2025
Elucidating the mechanisms underlying DNA double-strand break repair - PROJECT SUMMARY Our genomes are under constant threat due to the accumulation of DNA damage that arises from normal cellular metabolism and environmental hazards. DNA damage is a key driver of several human disorders, including neurodegeneration, cancer development, and progeroid syndromes. DNA double-strand breaks (DSBs) are the most deleterious type of DNA damage, which, if unrepaired, even a single DSB can initiate cellular dysfunction, tumorigenesis, and accelerated aging. DSBs are repaired via two distinct pathways: the high-fidelity homologous recombination pathway or the more error-prone non-homologous end-joining pathway. However, the critical molecular steps underlying the regulation of these pathways are still not completely understood. The long-term goal of my research program is to determine the underlying regulatory mechanisms of the multi-protein and multi- step process required to coordinate the repair of DNA damage. DNA resection is a key step in DNA repair pathway choice, which involves the degradation of the 5’ DNA strand of a DSB to form a long 3’ single-stranded DNA overhang used for homology-directed repair. This process requires the precise coordination of a dynamic multi-enzyme machine consisting of multiple nucleases, helicases, and regulatory proteins to degrade DNA along chromatin. However, a mechanistic description of how these enzymes assemble and are regulated at a single DNA break still needs to be discovered. This proposal aims to understand how this multi-enzyme complex comes together to coordinate its activities to prevent genome instability at the molecular level. In particular, we will answer fundamental questions regarding human genome maintenance, including: (1) How does DNA resection occur along chromatin? (2) How is DNA resection terminated (3) How is the DNA resection machinery utilized beyond DSB repair, such as at telomeres, to prevent genome instability? Deciphering these critical molecular events remains challenging because traditional approaches cannot directly observe the dynamic assembly of multiple DNA repair proteins on the same DNA molecule. We will use cutting-edge single-molecule fluorescence microscopy techniques, along with cryo-electron microscopy and biochemical techniques, to map the dynamics of DNA repair proteins at DSBs and telomeres. Together, this work will provide a better understanding of the complex spatial and temporal organization of key proteins during DNA repair and will undoubtedly provide new insights into how the accumulation of DNA damage results in the loss of genomic integrity, leading to tumor progression or accelerated aging syndromes.
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