Roles of Telomeric Oxidative DNA Lesions in Telomere Length Regulation

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The goals of this project are to determine how oxidative DNA damage regulates the homology-directed repair (HDR) pathways that enable telomere maintenance by the alternative lengthening of telomeres (ALT) mechanisms. Dysfunctional redox regulation is common among cancers and elevates reactive oxygen species, which generate DNA lesions. Telomeres are highly susceptible to oxidative damage but are essential for genome stability and sustained cell proliferation. Dysfunctional telomeres arrest cell division or drive excess chromosomal instability in pre-malignant cells that can cause cell death. To survive and achieve unlimited proliferation, cancer cells elongate and stabilize telomeres by activating telomerase or ALT. Although less common, ALT-driven cancers are highly aggressive with poor prognosis. Telomeric single-stranded overhangs are substrates for both telomerase and ALT-mediated telomere elongation but can self-fold into stable secondary structures. Previously, we found that low 8oxoG levels stimulate telomerase by altering structure and overhang accessibility, but that excess 8oxoG damage impairs telomere replication. In this project we will test the hypothesis that 8oxoG formation and processing at telomeres modulate ALT by promoting replication fork stalling and altering telomere structures. Using an innovative targeting tool to selectively induce 8oxoG at telomeres, we obtained preliminary data that 8oxoG increases numerous ALT hallmarks including C-circles, ALT-associated PML bodies, and telomeric sister chromatid exchanges and mitotic DNA synthesis. Aim 1 will determine how oxidative base damage modulates ALT and HDR activity in human cancer cell lines, proficient and deficient for repair. We will induce telomere specific damage or general oxidative stress in ALT and telomerase positive cells, and will measure cell survival, ALT phenotypes, and various telomere parameters. ALT requires RAD51 or RAD52 and is regulated by a variety of telomeric structures. Aim 2 will examine how oxidative lesions modulate telomeric RNA (TERRA) invasion and R-loop formation into telomeres. Using a single molecule fluorescence resonance energy transfer detection system, we discovered 8oxoG in telomeric duplex facilitates TERRA association. We will use complementary cellular studies to examine TERRA recruitment to telomeres after oxidative damage. Aim 3 will examine how oxidative damage in the telomere overhang modulates D-loop formation by RAD51-mediated strand invasion, or RAD52-mediated strand annealing, by using complementary single molecule and biochemical experiments. We will conduct cellular studies to determine how RAD51 or RAD52 deficiency influence 8oxoG-induced ALT phenotypes. This project will fill a significant void in our understanding of how general oxidative stress and 8oxoG, in particular, alter telomere restoration by homology-directed repair and ALT. Ultimately, this knowledge will be highly valuable for developing new strategies that 1) preserve telomeres to mitigate the effects of oxidative stress on healthy cells or conversely, that 2) inhibit telomere repair in malignant ALT tumors to halt proliferation.