Deciphering the mechanisms of PARP1 activity in telomere integrity The goals of this project are to define poly(ADP-ribose) polymerase 1 (PARP1) roles in telomere preservation under normal conditions and after environmental genotoxic exposure. My strong background in the PARP field and my ongoing training in telomere biology, place me in a unique position to successfully complete this project. In the long term, this project will lay the foundation for me to establish an independent research program investigating roles for PARPs in genome stability and human health. Telomeres consist of TTAGGG repeat arrays bound by the 6-member shelterin protein complex, and are lengthened by the reverse transcriptase telomerase in stem cells and most cancer cells. Telomere attrition and dysfunction can arise from exposure to various environmental agents and are associated with aging-related diseases and cancer. Previous studies have implicated PARP1 in telomere maintenance, but the mechanisms are poorly understood. PARP1 synthesizes poly(ADP-ribose) (PAR) and this activity is critical for genome maintenance by facilitating DNA repair pathways and regulating DNA replication during replication stress. The central hypothesis of this proposal is that PARP1 preserves telomere integrity by modulating the activities of the telomere shelterin proteins and telomerase. In support of this, I demonstrated that shelterin proteins as well as telomerase, bind to PAR. I also observed that PAR binding stimulates telomerase activity in vitro.
Aim 1 will define interactions between PARP1 and the various shelterin proteins under normal conditions and after genotoxic insult, and the roles for these interactions in telomeric DNA repair. We will test for PARylation of shelterin proteins, and will define their affinity for PAR. Oxidative and alkylating DNA damage will be induced with H2O2 and MNNG respectively, and 8-oxoguanine will be locally induced at telomeres using an innovative targeting system. We will follow PARP1 recruitment to telomeres after DNA damage, and will measure DNA repair rates and telomere aberrations in PARP1 proficient and deficient cells.
Aim 2 will examine PARP1 roles in regulating telomerase. We will examine PAR binding to reported putative PAR binding motifs (PBMs) in the hTERT subunit, and will examine the effect on telomerase activity by mutating these PBMs. We will monitor telomere length alterations in cells expressing wild type and PBM mutant hTERT, and telomerase recruitment to telomeres in PARP1 proficient and deficient cells.
Aim 3 will test PARP1 roles in telomere preservation upon replication stress induced with aphidicholin or G-quadruplex ligands. We will examine the recruitment of PARP1 to telomeres, and replication progression at telomeres in PARP1 proficient and deficient cells using the established single molecule analysis of replicated DNA (SMARD) assay. Completion of this project will uncover new interactions and relationships between PARP1 and the telomere specific proteins in preserving telomere integrity after DNA damage and replication stress. In the long term, this work will have important implications for the development of new cancer therapeutic strategies that target PARP1 functions in telomere maintenance.
PARP1 enzyme plays a central role in cellular responses to oxidative and DNA replication stress that can arise from genotoxic environmental exposures and lead to pathological conditions including inflammatory disease, features of accelerated aging and cancer. Telomeres at chromosome ends are highly susceptible to oxidative and replication stress. The goal of this proposal is to uncover mechanistic functions of PARP1 in preserving telomere integrity under normal and genotoxic conditions, which will aid in developing new therapeutic strategies for mitigating the effects of genotoxic environmental exposures on human health.
