DNA double-strand breaks (DSBs) are considered the most cytotoxic DNA lesions, often resulting in aberrant chromosomal translocations that promote carcinogenesis. In eukaryotes, DSB repair takes place in chromatin, a complex of DNA and histone proteins, and involves the two main pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). These pathways are regulated by a cascade of histone post-translational modifications that control the ordered assembly of DSB repair proteins. Central to this cascade is the RING-finger E3 ubiquitin ligase RNF168. By a mechanism that remains to be established, RNF168 and its cognate E2 ubiquitin-conjugating enzyme ubiquitylate chromatin specifically at lysine residues K13 and K15 of histones H2A and H2A.X (H2AK13ub and H2AK15ub). RNF168 is crucial for the recruitment of several downstream repair factors involved in regulating the balance between HR and NHEJ, but the underlying mechanisms linking RNF168 to these factors are unclear. We have two main objectives corresponding to two aims in this proposal.
In Aim 1, we will probe the recognition of the ubiquitylated nucleosome core particle (NCP) by a series of HR and NHEJ repair proteins using structural and cell biology approaches.
In Aim 2, we will explore the enzymatic mechanism of RNF168 and associated E2 ubiquitin-conjugating enzyme through structure determination and biochemistry. High-resolution structures of RNF168 complexes, as we propose, have the potential to reveal unprecedented details on the mechanism of action of a monomeric RING ubiquitin ligase. Collectively, in addition to providing a basic mechanistic understanding of an important DNA damage response enzyme, this research will help us understand how site specificity is conferred in both generating and reading chromatin ubiquitylation.
The proposed studies will help elucidate the basic molecular mechanisms of DNA repair ubiquitin ligase RNF168 and associated repair factors such as RAP80/BRCA1 important for cancer suppression. For example, mutations in RNF168 cause the cancer predisposing RIDDLE syndrome while mutations in RAP80/BRCA1 are linked to familial breast and ovarian cancer. The work has relevance to public health because understanding how RNF168 ubiquitylates chromatin and how these ubiquitylation signals are read and interpreted by DNA repair factors like RAP80/BRCA1 would help devise new therapeutic strategies for cancer treatment.
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