Repair of Double stranded DNA breaks-pathway choices and more
Double stranded DNA breaks (DSBs) are critical for cell health as a single unrepaired DSB is sufficient for inducing apoptosis. Two major mechanistically distinct pathways, homologous recombination (HR) and non-homologous end joining (NHEJ), have evolved to deal with DSBs and are regulated by factors that are conserved from yeast to mammals. The relative contribution of the competing DSB repair pathways differ in the different cell types and in different phases of the cell cycle, and this balance is critical for maintaining genomic stability.
Interplay of DSB repair pathways during cell cycle and impact on therapy: A decisive factor in the choice between DSB repair pathways is in the competition between DNA end protection (necessary for NHEJ) and DNA end resection (necessary for HR). DSB end resection must be appropriately restricted to S/G2 phases of the cell cycle, as HR requires the presence of an intact sister chromatid. Depletion of NHEJ promoting factors such as 53BP1 allows DNA end resection in the G1 phase, thereby impairing DSB repair and causing genomic instability. Conversely, loss of the HR protein, BRCA1 (critical for initiating end resection) allows the error-prone NHEJ pathway to dominate throughout the cell cycle potentially also contributing to mutations/deletions. From the perspective of therapy, loss of BRCA1 provides a therapeutic opportunity as these tumors are exquisitely sensitive to inhibitors of the DNA repair protein, poly (ADP-ribose) polymerase (PARP), and are also susceptible to platinum-based drugs. Surprisingly, loss of 53BP1 or associated factors (RIF1, PTIP) in these tumors render them insensitive to PARP inhibitors, as DNA end resection and the subsequent steps of the HR pathway is restored.
(a) MicroRNAs in DSB repair –pathway choice and therapeutic implications: We have conducted functional screens to discover microRNA (miRNA)s that down-modulate DSB repair proteins, and influence specific repair pathways. Ectopic expression of NHEJ-regulating miRNAs in BRCA1-deficient ovarian causes resistance to PARP inhibitors and platinum-drugs. In turn these patients have poor prognosis. We now utilize focused sequencing of miRNAs from BRCA deficient tumors that are resistant to therapy to identify miRNAs that influence the DNA repair machinery. Importantly these miRNAs have significant clinical relevance as therapeutic targets or predictive biomarkers of response.
(b) Systematic identification of factors that influence the BRCA-pathway: The (CRISPR)-Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines with disrupted genes. We are utilizing the whole-genome CRISPR library to comprehensively identify factors that restore HR-mediated DSB repair in BRCA1/2-deficient ovarian tumors and make these tumors resistant to PARP inhibitors and platinum-drugs. The goal is to better understand the biology of DSB repair, cross-talk with other signaling pathways and elucidate the mechanism of chemo-resistance.
(c) Non-coding (nc) RNAs and RNA binding proteins that impact DSB repair: Using cross-linking/immunoprecipitation and RNA-Seq we now have evidence that uncharacterized non-coding RNAs may be associated with DNA repair factors and directly impact the repair process. We are investigating the precise mechanism by which these ncRNAs associate with and functionally impact DSB repair.
Down-regulation of DSB repair in mitosis and activation via dephosphorylation: Interestingly in mitosis DSBs are recognized but not repaired, that is, factors like 53BP1 and BRCA1 are excluded from DSBs. Mitosis is the only phase of the cell cycle that lacks a DNA damage checkpoint.
(a) Why activating DSB repair pathways in mitosis leads to genomic instability? We have recently observed that phosphorylation of 53BP1 at specific residues during mitosis impedes its recruitment to chromatin and DSBs. Dephosphorylation via a PP4/R3beta phosphatase complex restores activity in the G1 phase. Counter-intuitively we observe that ectopic activation of the 53BP1 pathway, consequently the NHEJ pathway, in mitotic cells causes genomic instability and mitotic defects. We are addressing this question using a combination of cytological tools and single-cell sequencing.
(b) Phosphatases in DSB repair. We have demonstrated that phosphatases participate in multiple steps of the DNA damage response, which includes facilitating DNA repair in the context of the cell cycle phase, restoration of chromatin structure and regulating checkpoints. We now have evidence that dephosphorylation of specific phospho-residues of DNA repair proteins is a pre-requisite for their function. We have developed a novel phospho-proteomic strategy to identify these factors and also to identify phosphatases that regulate their activity. Our goal is to continue these studies and systematically investigate the role of phosphatases in DSB repair.
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