Structural and Biological Effects of Ribonucleotide Insertion into Telomeres

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Project Abstract Telomeres are protective noncoding DNA caps at the ends of chromosomes that maintain genomic integrity. In most somatic tissues telomeres will shorten with successive rounds of replication and will reach a critically short length, at which point a cell will become senescent or undergo apoptosis. This fate can be avoided if telomeres are maintained through expression of telomerase, a reverse transcriptase that elongates telomeres by adding telomeric repeats. This elongation of telomeres can lead to replicative immortality, one of the hallmarks of cancer. Another hallmark of cancer is genomic instability that arises from DNA damage. The most prevalent form of DNA damage are ribonucleotides (rNTPs) inserted during DNA replication. Left unrepaired these rNTPs will promote genomic instability, changes to the DNA secondary structure, and human diseases. Given the deleterious effects of rNTPs in DNA, cells have evolved the ribonucleotide excision repair (RER) pathway to remove rNTPs. While the impact of rNTPs in the genome is well established to have deleterious effects and promote human disease, the impact of rNTPs at telomeres remains unknown. One essential DNA secondary structure seen at telomeres, that can be affected by rNTPs, is a G-quadruplex (G4). Specific to this proposal, it is not known what effect rNTPs will have on telomeric structure and integrity, or how rNTPs at telomeres are repaired to protect telomere integrity. To investigate this, we have developed a telomerase mutant (Y717A) that inserts rNTPs at a greater rate than wildtype (WT) telomerase. Using this mutant, I will selectively increase the rate of rNTP incorporation only at telomeres. The overarching goal of this proposal is to determine the effect of rNTPs on telomeric G4s and characterize the repair pathway for rNTPs in telomeres. I hypothesize that rNTP insertion into telomeres will alter telomeric G4 dynamics and that these rNTPs are repaired through RER. To test this hypothesis, I propose the following specific aims: 1) characterize the structural effects of rNTP insertion into telomeres and 2) characterize rNTP repair in telomeres. For aim 1, I will use circular dichroism spectrophotometry to systematically examine G4 formation in vitro. Using oligos of the basic telomeric sequence that will form a G4 (TTAGGG)4, I will systematically replace the dNTPs with rNTPs to determine if rNTPs alter the key G4 structural motif in telomeres. Additionally, using cell lines that contain either WT telomerase or a telomerase mutant which inserts rNTPs at a greater rate than WT in conjunction with antibodies that are specific for G4s to examine if the presence of rNTPs alters the G4 content at telomeres. For aim 2, I will characterize RER by reconstituting the RER pathway with a substrate that has a single rNTP that will either be linear DNA or DNA folded into a G4. I will monitor the formation of the repaired product and compare RER functionality on the different substrates. Additionally, using proximity dependent biotin identification (BioID) I will be able to identify any additional proteins that are involved in repair of rNTPs at telomeres. This study applies an innovative combination of in vitro biochemical assays with cellular assays to examine the impact and repair of rNTPs at telomeres.