Telomeres protect chromosome ends from being repaired as double-strand breaks (DSBs). Just as DSB repair is suppressed at telomeres, de novo telomere addition is suppressed at the site of DSBs. To identify factors responsible for this suppression, we developed an assay to monitor de novo telomere formation in Drosophila, an organism in which telomeres can be established on chromosome ends with essentially any sequence. Germline expression of the I-SceI endonuclease resulted in precise telomere formation at its cut site with high efficiency. Using this assay, we quantified the frequency of telomere formation in different genetic backgrounds with known or possible defects in DNA damage repair. We showed that disruption of DSB repair factors (Rad51 or DNA ligase IV) or DSB sensing factors (ATRIP or MDC1) resulted in more efficient telomere formation. Interestingly, partial disruption of factors that normally regulate telomere protection (ATM or NBS) also led to higher frequencies of telomere formation, suggesting that these proteins have opposing roles in telomere maintenance vs. establishment. In the ku70 mutant background, telomere establishment was preceded by excessive degradation of DSB ends, which were stabilized upon telomere formation. Most strikingly, the removal of ATRIP caused a dramatic increase in telomeric retrotransposon attachment to broken ends. Our study identifies several pathways thatsuppress telomere addition at DSBs, paving the way for future mechanistic studies.T ELOMERES are nucleoprotein structures that serve two vital functions. First, they overcome chromosome end shortening by regulating the addition of new sequences. Second, they prevent chromosome ends from being recognized as double-strand breaks (DSBs). Failure in this second function (the capping function) results in fusions between telomere ends and subsequently in genomic instability. Just as DSB repair is suppressed at the telomere, de novo telomere establishment is suppressed at the site of DSBs. Telomere healing, the addition of telomere functions at a DSB, stabilizes the broken chromosome but leads to loss of heterozygosity due to aneuploidy. Understanding the process of de novo telomere formation will shed light on the cellular mechanisms that normally prevent aneuploidy formation for genome maintenance. In particular, primordial germ cells might lack the apoptotic response elicited by the presence of persistent DSBs possibly due to their quiescent state during early development (Hanyu-Nakamura et al. 2004;Renault et al. 2004;Sano et al. 2005). In these cells, unrepaired breaks might be allowed to persist long enough to acquire telomeres increasing the potential of forming aneuploid cells. Telomere healing can result in human diseases (Flint et al. 1994;Varley et al. 2000;Bonaglia et al. 2011).In most eukaryotic organisms studied, telomere elongation is carried out by the telomerase enzyme. In these systems, telomere formation on DSBs depends on telomerase function to add short repeats to broken ends. However, it rema...
Telomeres are obligatory chromosomal landmarks that demarcate the ends of linear chromosomes to distinguish them from broken ends and can also serve to organize the genome. In both budding and fission yeast, they cluster at the periphery of the nucleus, potentially to establish a compartment of silent chromatin. To gain insight into telomere organization in higher organisms, we investigated their distribution in interphase nuclei of Drosophila melanogaster. We focused on the syncytial blastoderm, an excellent developmental stage for live imaging due to the synchronous division of the nuclei at this time. We followed the EGFP-labeled telomeric protein HOAP in vivo and found that the 16 telomeres yield four to six foci per nucleus, indicative of clustering. Furthermore, we confirmed clustering in other somatic tissues. Importantly, we observed that HOAP signal intensity in the clusters increases in interphase, potentially due to loading of HOAP to newly replicated telomeres. To determine the rules governing clustering, we used in vivo imaging and fluorescence in situ hybridization to test several predictions. First, we inspected mutant embryos that develop as haploids and found that clustering is not mediated by associations between homologs. Second, we probed specifically for a telomere of novel sequence and found strong evidence against DNA sequence identity and homology as critical factors. Third, we ruled out predominance of intrachromosomal interactions by marking both ends of a chromosome. Based on these results, we propose that clustering is independent of sequence and is likely maintained by an as yet undetermined factor.
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