Specific Aim 1: Determine the functional significance of 53BP1 DNA binding.
- Aim 1A: 53BP1 DNA binding mutant construction and characterization.
- Aim 1B: Elucidate the role of 53BP1 DNA binding in DSB repair pathway choice and replication fork protection.
Specific Aim 2: Understand the antagonistic roles of CST and BRCA1-BARD1 in EXO1 regulation.
- Aim 2A Delineate the mechanism through which CST inhibits EXO1 nuclease activity.
- Aim 2B Determine the mechanism by which BRCA1-BARD1 overcomes the CST blockade of EXO1.
- Aim 2C Single-molecule DNA curtain analyses of EXO1 regulation by BRCA1-BARD1 and CST.
Specific Aim 3: Understand the antagonistic roles of CST and BRCA1-BARD1 in BLM-DNA2 regulation.
- Aim 3A. Delineate the mechanism by which CST restricts end resection by BLM-DNA2.
- Aim 3B. Interrogate BRCA1-BARD1 for the ability to alleviate the CST blockade.
- Aim 3C. Single-molecule DNA curtain analysis of end resection by BLM-DNA2 and regulation by CST.
Abstract
DNA double strand breaks (DSBs) are induced by genotoxic agents such as ionizing radiation, chemotherapeutic agents, and during encounters of the DNA replication machinery with DNA damage. The two major, mechanistically distinct DSB repair pathways are non-homologous DNA end joining (NHEJ) and DNA homology-directed repair (HDR). NHEJ is efficient but error-prone. HDR is inherently accurate and represents the preferred repair tool for DNA replication-associated DSBs. HDR commences with the resection of the 5’-terminated strand at break ends to generate a DNA tail that serves as the template for the assembly of the RAD51 recombinase filament. DSB repair pathway choice is linked to cell cycle progression and is determined by whether or not a DSB undergoes extensive resection. Long-range resection is principally mediated by the 5’-3’ exonuclease EXO1 or the BLM helicase- DNA2 endonuclease. The chromatin reader 53BP1 nucleates the formation of a higher-order ensemble that harbors the CTC1-STN1-TEN1 (CST) complex at DSB ends to block end resection in the G1 phase of the cell cycle. The restrictive action of the 53BP1 axis is alleviated by BRCA1-BARD1 in the S and G2 phases via mechanisms that are poorly understood. Thus, BRCA1-deficient tumors, on account of their HDR deficiency, are particularly vulnerable to PARP inhibitors (PARPi) due to synthetic lethality. However, dysfunction in the 53BP1 axis leads to HDR restoration and PARPi resistance.
We have discovered that 53BP1 binds double-stranded DNA. We hypothesize that DNA binding by 53BP1 is germane for its role in the recruitment of downstream factors and the imposition of DSB repair pathway choice. Importantly, we find that the CST complex physically interacts with and inhibits EXO1 as well as BLM-DNA2, while BRCA1-BARD1 efficiently overcomes these restrictive activities of CST. We hypothesize that CST acts via physical interaction with resection enzymes and association with ssDNA to attenuate DNA end resection, while BRCA1-BARD1 counters CST by disrupting these inhibitory interactions and by stimulating resection enzymes. To elucidate the underpinnings of the DNA end resection restriction circuitry, we will (1) determine the biological role of DNA binding by 53BP1, (2) delineate how CST interferes with the activity of the 5’-3’ exonuclease EXO1 and of the helicase-endonuclease complex BLM-DNA2 in DNA end resection, and (3) interrogate BRCA1-BARD1 for its ability to overcome the restriction of DNA end resection imposed by CST.
Our studies and synergy with Projects 1 and 3 will elucidate the intricate regulatory networks that control the onset of DNA end resection and efficiency. These collaborative endeavors will not only illuminate the mechanistic principles of DSB repair pathway choice but will also exert a major impact on our understanding of how DSBs and stressed DNA replication forks lead to cancer and will provide actionable information to help guide the development of targeted cancer therapies to treat BRCA-deficient cancers.
