Sunday, May 18, 2025
5/18/2025
Ribonucleotide Processing in Telomere Maintenance and Integrity - Abstract Chromosomal ends harbor repetitive DNA sequences, termed telomeres, that are recognized by the shelterin complex to form a lariat structure called the T-loop, which protects vital genetic information and prevents erro- neous DNA double strand break repair. Each cell division these telomeres are shortened until they reach a crit- ically short length, causing cellular apoptosis or senescence. This shortening can be counteracted by the ribo- nucleoprotein telomerase, which functions to maintain telomere integrity by elongating the 3’ end of telomeres. Because of this fundamental role played by telomerase, about 85 - 90% of all cancers upregulate telomerase to lengthen telomeres and achieve immortality. Therefore, understanding telomere biology and how telomerase maintains telomere ends is vital for understanding carcinogenesis. One such telomeric change is the insertion of ribonucleotides (ribos) in telomeres by telomerase. Ribos represent the most prevalent form of genomic DNA damage. Unrepaired genomic ribos are linked to multiple types of cancer and are a source of PARP-trap- ping lesions which can compromise genome stability. Similar to genomic ribos, ribos inserted by telomerase at telomeres are highly susceptible to spontaneous hydrolysis and may promote premature telomere shortening through strand breakage. However, the biological impact of ribos within telomeres has been unexplored. This is, in part, because introducing ribos at telomeres without altering the rest of the genome has not been possi- ble. We have overcome this limitation by developing a telomerase mutant that is highly enhanced for ribo inser- tion and identified a cancer associated telomerase mutation that is moderately enhanced for ribo insertion at telomeres. We hypothesize that ribos are present at telomeres and impact telomere integrity, destabilize telo- mere architecture, and activate ribonucleotide excision repair. To address this, our collaborative team pro- poses cellular, biochemical, single-molecule, structural, genome ribo mapping, and proteomic approaches. In Aim 1, we will investigate the levels of ribos in telomeres, the impact of ribos on telomere integrity and cellular viability, and how ribos in telomeres alter the DNA damage response at telomere ends. In Aim 2, we will deter- mine how telomeric ribos alter the binding of the shelterin complex to telomeric DNA and how ribos alter the formation, stability, and telomerase dependent resolution of G-quadruplexes. In Aim 3, we will investigate how ribos within telomeres are identified, removed, and repaired by the ribonucleotide excision repair pathway. This application is relevant to cancer because: (1) changes in telomere integrity are associated with an increased risk of cancer; (2) the presence of unrepaired ribos are linked to cancer; (3) there is a significant gap in our un- derstanding of the impact of ribos on telomere integrity; (4) ribos likely promote telomere instability, which would induce extensive chromosomal instability and drive malignant transformation in surviving cells; (5) we are characterizing a cancer-associated variant with enhanced ribo insertion; and (6) understanding how ribos alter telomere integrity and maintenance will aid in developing strategies to protect telomere integrity.
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