Monday, May 29, 2023
5/29/2023
Project Summary DNA double stranded breaks (DSBs) interfere with cellular viability, but also initiate chromosomal translocations resulting in genomic instability and promoting carcinogenesis. BRCA1 and BRCA2 proteins are essential for homologous recombination (HR)-mediated repair of DSBs. Understanding the mechanisms of the BRCA pathway has broad implications for human health. When replication forks encounter damaged DNA, they arrest and unless properly processed, they collapse leading to DNA breaks and genomic instability. To avoid their collapse, stalled forks can be reversed by annealing the two nascent strands to each other, in a process catalyzed by DNA translocases such as ZRANB3. The BRCA proteins load RAD51 on reversed forks to protect the DNA ends against degradation by the nuclease MRE11. The ability to protect forks against degradation corelates with DNA damage sensitivity. Thus, replication fork protection is essential for DNA repair and genomic stability. However, how protection of stalled replication forks against nucleolytic degradation is achieved represents a major knowledge gap. The PARP family at least 17 members, with various and lesser understood functions than the founding member PARP1. PARP14 has been associated with multiple cellular processes, but mechanistic details are generally sparse. We previously showed that PARP14 loss reduces HR efficiency and sensitizes cells to radiation. Recently, we have identified a novel role of PARP14 in promoting replication fork degradation, genomic instability and DNA damage sensitivity, which is the focus on this application. For this application, our goal is to understand how PARP14 promotes fork degradation, resulting in DNA damage sensitivity of BRCA-deficient cells. Our overall hypothesis is that PARP14 interferes with the RAD51-MRE11 mechanism of control of DNA resection at reversed replication forks to trigger nascent strand degradation, thus enhancing DNA damage sensitivity in BRCA-deficient cells. Aim 1 is to reveal the impact of PARP14 on RAD51-mediated protection of stalled replication forks. We hypothesize that PARP14 interferes with BRCA-independent stabilization of RAD51 on reversed forks, to enhance their degradation. Aim 2 is to uncover how PARP14 engages MRE11 for nucleolytic degradation of damaged forks. We hypothesize that PARP14 binds to stalled replication forks in BRCA-deficient cells and recruits MRE11 to initiate nucleolytic degradation of nascent DNA at these structures. Aim 3 is to elucidate the role of KU in fork protection against nucleolytic resection by EXO1 and MRE11. We hypothesize that KU binding to reversed forks protects them against EXO1-mediated degradation, but enables nascent strand resection by the MRE11-PARP14 complex. Since DNA damaging agents promote genomic instability by inducing nascent strand degradation, potentially underlying their carcinogenesis, successful accomplishment of these Specific Aims would reveal a new mechanism of genome stability and tumor suppression, centered on PARP14. It may also reveal PARP14 as a biomarker for the tumor response to radiation and genotoxic chemotherapy, in the context of the BRCA status.
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