Determining the effects of ribonucleotides on telomere integrity

点击翻译按钮获取中文摘要

Project Summary/Abstract Ribonucleotides (ribos) are misinserted by DNA polymerases and have recently been recognized as the most common lesion across most of the genome. Compared to deoxyribonucleotides, ribos have an additional 2'-OH that makes them much more susceptible to spontaneous hydrolysis. Additionally, unrepaired ribos promote genomic instability and are associated with various human diseases. In comparison to genomic ribos, the impact of ribos in telomeres is unknown. Telomeres are protective noncoding DNA structures at the ends of chromosomes that are essential for maintaining genomic integrity, as highlighted by the role of telomere dysfunction in a myriad of human diseases. Due to the lack of a tool to selectively perturb ribo insertion rates into telomeres, the prevalence, and effects of telomeric ribos remains unexplored. To this end, we have developed solutions for investigating the role of telomeric ribos by discovering a variant telomerase that lacks any ribo discrimination activity and by developing an assay to determine the levels of telomeric ribos. Furthermore, I have identified two telomerase disease associated variants (A716V and Y717N) which appear to perturb telomerase ribo discrimination. The overarching goal of this proposal is to determine the levels and effects of telomeric ribos, as well as establish a causal role for increased telomeric ribo insertion and human disease. I hypothesize that ribonucleotides are present in telomeres and that increased ribo insertion promotes telomere uncapping and genomic instability. To test this, I propose the following aims: 1) Characterize the kinetic and structural effects of telomerase disease-associated variants and 2) Determine how each variant effects telomere integrity and cell viability. To accomplish these aims I will characterize the pre-steady state kinetics of A716V and Y717N using the telomerase homolog Tribolium castaneum telomerase reverse transcriptase (tcTERT) (Aim 1A), as well as use human telomerase to determine differences in telomerase processivity (Aim 1B). I will complement the kinetic analysis by characterizing the structural rearrangements of A716V and Y717N in tcTERT using X-ray crystallography (Aim 1C). Following the comprehensive biochemical characterization of each variant, I will determine the levels of telomeric ribos in cell lines expressing each telomerase variant (Aim 2A). Finally, the effects of increased telomeric ribos will be determined using immunofluorescence microscopy to identify telomere dysfunction induced foci (Aim 2B), which are a commonly used marker of a telomere DNA damage response. This innovative project will be completed at the University of Kansas Medical Center under the supervision of an expert sponsorship team. In addition to technical skills, the training plan also prioritizes training in scientific communication and mentorship. The proposed experiments will provide me with the foundation for a successful post-doctoral fellowship and, ultimately, a productive independent research career.