Progressive telomere shortening, which naturally occurs over several cell cycles or more rapidly due to telomere dysfunction, eventually causes cells to undergo senescence. Telomeres are the protective nucleoprotein structures at the end of linear chromosomes that represent the replicative lifespan of a cell and must be maintained for cell viability. One way immortal cells, such as germ and certain stem cells, ensure sufficient telomere length is by having the telomere-specific reverse transcriptase telomerase active. Most somatic cells repress telomerase and therefore must solely rely on proteins that protect the telomeric ends for viability. An important protein in this regard is the Ku heterodimer, a high affinity DNA end-binding protein known for its critical roles in nonhomologous end joining (NHEJ) and telomere structure and maintenance. Ku impacts several aspects of telomere biology including regulating telomere length by recruitment of telomerase and also protecting telomeric ends from exonucleolytic degradation and recombination, a property known as telomere end protection. Recently, the sponsor and co-workers have generated DNA end-binding defective Ku mutants that exhibit global telomere dysfunction, including accelerated senescence in the absence of telomerase. These data strongly suggest that Ku must directly bind the telomeric end to perform its telomeric functions. How Ku is recruited to telomeres and accesses the end remains unclear. One mechanism of recruitment may be through chromatin remodeling complexes that have been shown to recruit Ku to double strand breaks. The overall goal of the proposed work is to determine which factors affect Ku's ability to associate with telomeric ends, thereby affecting telomere maintenance.
Specific Aim 1 will use a biochemical approach to determine whether Ku's association with the RNA subunit of telomerase, TLC1, influences its ability to bind a telomeric DNA end and vice versa, and site directed mutagenesis to determine the binding site on Ku for TLC1.
Specific Aim 2 will characterize the influence of ATP-dependent chromatin remodeling complexes on Ku's localization at the telomeric end using genetic and molecular approaches.
Specific Aim 3 will extend these analyses to human cells, where Ku is essential, via characterizing Ku's association with telomeres throughout the cell cycle and in the absence of possible recruiting proteins or its DNA ending activity. How the absence of Ku's DNA end binding activity impacts on telomere integrity will be determined. Ku deficient mice exhibit premature aging, which may relate to Ku's role in maintaining telomere structure and function. Thus, the studies proposed may take us one step further to understanding the mechanism by which Ku suppresses aging, and may ultimately lead to insight into aging-related diseases.
Through a combination of genetic and molecular techniques, the proposed studies will provide important insight into how chromosome ends are maintained. The studies will be conducted in the Baker's yeast, which is a powerful model system to study chromosome ends as well as in human cells. The work has the potential to provide insight into mechanisms that contribute cellular aging.
|Ribes-Zamora, Albert; Indiviglio, Sandra M; Mihalek, Ivana et al. (2013) TRF2 interaction with Ku heterotetramerization interface gives insight into c-NHEJ prevention at human telomeres. Cell Rep 5:194-206|
|Lopez, Christopher R; Ribes-Zamora, Albert; Indiviglio, Sandra M et al. (2011) Ku must load directly onto the chromosome end in order to mediate its telomeric functions. PLoS Genet 7:e1002233|