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Structural and Biological Effects of Ribonucleotide Insertion into Telomeres
Cortez, Luis Manuel
Cortez, Luis Manuel
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Abstract
Telomeres are structures that protect the ends of chromosomes to preserve genomic integrity. Telomeres are maintained by telomerase, which adds telomeric repeats (TTAGGG) to the ends of chromosomes. Telomeres are composed of deoxyribonucleotide monophosphates; however, telomerase has been shown to insert ribonucleotide monophosphates as efficiently as replicative DNA polymerases. Non-telomeric ribonucleotide insertions are deleterious due to the additional hydroxyl group altering DNA structure and causing strand breakage. However, the effect on telomeres remains poorly explored. We hypothesized that ribonucleotide insertion at telomeres would have deleterious consequences. Here we determined the deleterious effects on ribonucleotides on telomeres. Using two mutant variants of the catalytic subunit of telomerase which insert ribonucleotides at a greater rate than wildtype, we were able to selectively introduce ribonucleotides at telomeres to examine their effects. We found that in cancer cells, specifically the cervical cancer HeLa cell line and the colorectal cancer HCT116 cell line, insertion of ribonucleotides leads to decreases in telomere length as well as an increase in telomeric instability and the formation of micronuclei. Additionally, we saw an increase in the DNA damage response (DDR) signaling in the HeLa cell line upon induction of ribonucleotides. In the retinal pigment epithelial (RPE) primary cells we see a similar increase in telomeric instability, formation of micronuclei and DDR response. We also provide evidence that ribonucleotides at telomeres are repaired through the ribonucleotide excision repair pathway. Given that the telomeres can form G-quadruplexes (G4s), as telomeres are G rich, we examined the effects of ribonucleotides on the formation of G4s. We found that ribonucleotide substitution of the first dG in a repeat (TTAGGG)4 altered the G4 conformation. Additionally, ribonucleotide substitution of the first or second dG in a repeat decreased thermal stability, and the decrease in thermal stability is positively correlated with increases in molecules exhibiting conformational dynamics. Additionally, RNase H2, the initiator of ribonucleotide excision repair, had reduced cleavage of ribonucleotides in G4s. Continuing our examination of telomeric integrity, we examined known variants of the catalytic subunit of telomerase, TERT, that lead to telomere biology disorders. We investigated the mechanism of how these disease associated TERT variants induce their effects. We used single-turnover kinetics and computer simulations to characterize the nucleotide insertion mechanisms of six disease-associated variants.
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Date
2025-01-01
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University of Kansas
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Cortez_ku_0099D_19974.pdf
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- Embargoed until 2176-05-31
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Biochemistry, Cellular biology, Molecular biology