In mammalian tissues, a hallmark of cellular aging is the loss of DNA sequence from chromosome ends with every cell division, due largely to telomere end resection, which generates single-stranded 3'overhangs at telomeres. Terminal sequence loss in normal cells can eventually lead to cell death or senescence, whereas cancer cells can circumvent this process. Our long-term goal is to lend insight into the biology of aging by identifying the as yet unknown factors involved in telomere end resection and characterizing their roles in determining cellular lifespan. Our research plan is based on the hypothesis that post-replicative telomere resection recapitulates the process of 5'end resection during DNA double strand break (DSB) repair. We will address our hypothesis through the following specific aims: 1) elucidate the mechanism by which mouse POTIb protects telomeres from excessive resection, 2) determine whether the mammalian orthologs of the factors involved in DSB repair in yeast contribute to telomere end resection, and 3) characterize the impact of telomere end resection on telomere attrition in normal and cancer cells. First, we will assess whether P0T1 b-deficient mouse cells, which undergo excessive end resection, can be used to dissect the physiologic mechanism of telomere end resection. Specifically, we will determine whether POTIb regulates the generation of the telomeric overhang in S phase. Next, we will analyze the telomeric overhang after deleting or knocking down candidate end resection factors in mammalian cells. We will perform similar genetic manipulations in cells from which POTIb can be deleted to determine whether depleting these factors prevents the aberrant resection phenotype that occurs in the absence of POTIb. Finally, we will determine the effect of depleting or overexpressing these factors on the rate of telomere shortening over successive cell divisions. In these studies, we will use previously developed methods to monitor the single-stranded telomeric overhang and telomere length. Our work to identify factors that govern cellular lifespan will open new avenues of investigation for diseases of the aging population, a growing public health challenge. On one hand, antagonizing these factors in aging tissues may delay the detrimental effects of cellular senescence. On the other hand, stimulating these factors to limit the proliferative capacity of cancer cells may offer improved options for cancer therapy.
|Wu, Peng; Takai, Hiroyuki; de Lange, Titia (2012) Telomeric 3' overhangs derive from resection by Exo1 and Apollo and fill-in by POT1b-associated CST. Cell 150:39-52|
|Wu, Peng; van Overbeek, Megan; Rooney, Sean et al. (2010) Apollo contributes to G overhang maintenance and protects leading-end telomeres. Mol Cell 39:606-17|