The synthesis and turnover of poly(ADP-ribose) (PAR) in response to DNA damage plays an important role in DNA damage repair. Notably, inhibitors of poly(ADP-ribose) polymerases (PARP) were developed to selectively target cancer cells with defects in homologous recombination.In addition, PARP-dependent repair of DNA single strand breaks (SSB)s is a major determinant in cell survival following exposure to genotoxic antitumor agents such as temozolomide and camptothecin. Recently, PARP-dependent repair of DNA double strand breaks (DSB)s by an alternative non-homologous end-joining (alt-NHEJ) pathway has been recently identified as a novel therapeutic target in therapy-resistant forms of breast cancer and leukemia. Progress by investigators during SBDR-3 was the driving force for the development of this new project as their research converged on a common theme of PARP-dependent responses to DNA damage. These efforts were supported by the development of new protein expression strategies with the EMB core and new small angle x-ray scattering approaches in the SCB core that have enabled us to gain insights into the size, shape and flexibility of key proteins complexes involved in PARP-dependent DNA repair. To enhance this project, we recruited a new investigator, Dr. Pascal, who has performed groundbreaking studies on the mechanism of PARP activation by DNA damage using structural and biochemical approaches. In this project, we will utilize the complementary strengths of our team in biophysics, biochemistry, structural and cell biology, and DNA repair inhibitor development to gain insights at the molecular level into the regulation of DNA damage- dependent PAR synthesis and turnover and the PARP-dependent repair of DNA strand-breaks.
In Aim 1, we will focus on the interplay between PARP-1 and PARG that regulates DNA damage- dependent PAR synthesis and degradation using structure-guided mutants that modulate the allosteric activation of PARP-1 and inhibitors of PARG to determine how changing the levels of PAR synthesis and NAD+ depletion affects DNA repair and cell viability.
In Aim 2, we will investigate how interactions between PARP-1 and XRCC1 complexes coordinate the repair of SSBs.
In Aim 3, we will elucidate the mechanisms of PARP- dependent repair of DSBs by alt-NHEJ, focusing on the interplay between XRCC1 and the hMre11/hRad50/Nbs1 complex and the assembly of alt-NHEJ factors at DSBs in collaboration with project 5. We envision that our studies will significantly advance understanding of the PARP-dependent responses to DNA damage and will guide the development of more effective therapies for cancer. Our studies will continue to make extensive use of the EMB and SCB cores and are highly integrated with the other four projects of SBDR-4. In particular, the role of PAR synthesis and turnover at the replication fork, in other DNA repair pathways and in repair pathway choice will be explored in collaboration with projects 1, 2, 3 and 5.
Project 4 ? PARP-dependent break repair PROJECT NARRATIVE The promising clinical results with the existing poly(ADP-ribose) polymerase (PARP) inhibitors provides a compelling rationale for the development of more selective PARP inhibitors and novel approaches to manipulating PARP-mediated responses to DNA damage. In this project, we will examine the effects of altering the levels of DNA damage-dependent poly (ADP-ribose) synthesis on DNA repair and cell viability, and determine the molecular mechanisms of a PARP-dependent repair pathway that plays an important role in resistance to chemotherapeutics and another PARP-dependent repair pathway that appears to be a cancer cell-specific target in therapy-resistant cancers. The overall goal of these studies is to provide a framework for the development of improved treatment strategies for cancer.
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