Thursday, April 3, 2025
4/3/2025
Molecular regulation of double-strand break repair by MDC1 - Project Summary: DNA double strand breaks (DSB) pose harmful threats to genomic integrity. For this reason, cells evolved a diverse set of tightly regulated pathways dedicated to repairing these lesions. In human cells, two major pathways are responsible for repairing most DSBs, including Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ). Mediator of DNA Damage Checkpoint 1 (MDC1) is a key DSBR protein that facilitates HR and NHEJ by binding to γH2AX at DSB sites and promoting RNF8- and RNF168-dependent ubiquitination of H2A at K13 and K15. Considering that MDC1’s functions in HR do not require BRCA1 and its deletion only mildly increases cellular sensitivity to DSBs, the exact mechanism by which MDC1 promotes HR has remained unclear. Using live-cell single-molecule (LCSM) imaging, we recently found that MDC1 is constitutively tethered to chromatin by its intrinsically disordered PST repeat domain, which has unknown functions in DSBR. Compelling preliminary data suggests this domain is essential for MDC1-dependent HR and is dispensable for MDC1- dependent NHEJ. Interestingly, LCSM imaging studies reveal the PST domain may spatially restrict MDC1- dependent HR by directly tethering MDC1 to transcriptionally active chromatin. In addition, our data demonstrate the PST-chromatin interaction can be negatively regulated by phosphorylation, which may link phospho- dependent changes in the PST-chromatin interaction with MDC1’s distinct functions in HR and NHEJ. Therefore, I hypothesize that MDC1-dependent HR is a specialized pathway dedicated to resolving transcription-associated DSBs that depends upon a regulatable interaction of MDC1’s PST domain with chromatin. To test this hypothesis, I will integrate a variety of molecular, biochemical and biophysical approaches with training in ChIP- seq and genome-wide CRISPR screening during the K99 phase of this award to: 1. Identify the molecular mechanism underlying MDC1’s PST-dependent association with chromatin (Aim 1); 2. Elucidate a functional role for MDC1 and its PST domain in transcription-coupled HR (Aim 2); and 3. Define the molecular mechanism of the MDC1-dependent HR pathway (Aim 3). Not only will the Aim 1 and 2 studies uncover the molecular mechanism of MDC1’s PST domain in DSBR, but they will also lead to the identification of MDC1 mutants with pathway-specific DSBR functions. Therefore, in Aim 3 I will use these pathway-specific MDC1 alleles and genome-wide CRISPR screens to genetically define the MDC1-dependent HR pathway. These studies will significantly advance our understanding of the fundamental processes by which cells maintain their genomic integrity by revealing the mechanistic basis for MDC1’s functions in homologous recombination. In addition to the mentored scientific and professional development opportunities provided during the K99 phase, results from the proposed studies will be used as preliminary data for R-series grants to be submitted during the R00 phase of this award.
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