Hotspots of de novo telomere addition as mediators of genomic instability in yeast

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Project Summary Telomeres are highly repetitive, protein-bound, G-rich sequences that cap the ends of most eukaryotic chro- mosomes. As substrates for the ribonucleoprotein complex telomerase, telomeres provide a mechanism to counteract the progressive loss of terminal DNA that occurs during replication. Telomeres also distinguish normal chromosome ends from broken ends in need of repair. Inappropriate recruitment of repair proteins to normal chromosome ends has disastrous consequences for cell viability by allowing formation of dicentric chromosomes as a result of end fusion events. Telomerase has the ability to create a new, functional telomere at an internal site following a double-strand break (DSB). Such de novo telomere addition can interfere with normal repair through the creation of terminal deletions. Indeed, chromosome truncations, some of which arise through de novo telomere addition, are a common source of human genetic disease. Given these consequences, it is not surprising that cells have evolved mechanisms to inhibit the action of telomerase at DSBs. Work recently published from our laboratory has characterized two sequences in the yeast genome that support much higher frequencies of de novo telo- mere addition than neighboring regions [termed SIRTAs (Sites of Internal Repair-associated Telomere Addi- tion)]. We hypothesize that SiRTAs are refractory to mechanisms that normally inhibit telomerase action at a DSB due either to specific cis- and trans-acting factors and/or because telomerase action is no longer inhibited when a break has persisted without repair. Here we characterize factors that contribute to the high frequency of telomere addition at SiRTAs and examine the regulation of telomerase action at these hotspots of genomic instability. Our experiments are unique in addressing the regulation of telomere addition at endogenous sites that may be located hundreds or thousands of nucleotides from the initiating DSB. Although the detrimental consequences of de novo telomere addition are clear, the distribution of genomic sites with abnormally high potential to mediate such events has not been determined in any organism. In her groundbreaking work in the 1940s, Barbara McClintock was the first to suggest that telomere addition could serve to cap a chromosome break, thus preventing chromosome loss in the face of otherwise irreparable dam- age. It is currently unclear whether genomic hotspots of telomere addition are a byproduct of selection for other functions, or may have evolved or been retained as a mechanism of DNA damage tolerance. The systematic identification of SiRTAs in yeast will provide tools to answer this question while highlighting the features that lead to genomic instability through de novo telomere addition.