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Telomere healing occurs when telomerase, normally restricted to chromosome ends, acts upon a double-strand break to create a new, functional telomere. De novo telomere addition (dnTA) on the centromere-proximal side of a break truncates the chromosome but, by blocking resection, may allow the cell to survive an otherwise lethal event. We previously identified several sequences in the baker's yeast, Saccharomyces cerevisiae, that act as hotspots of dnTA [termed Sites of Repair-associated Telomere Addition (SiRTAs)], but the distribution and functional relevance of SiRTAs is unclear. Here, we describe a high-throughput sequencing method to measure the frequency and location of telomere addition within sequences of interest. Combining this methodology with a computational algorithm that identifies SiRTA sequence motifs, we generate the first comprehensive map of telomere-addition hotspots in yeast. Putative SiRTAs are strongly enriched in subtelomeric regions where they may facilitate formation of a new telomere following catastrophic telomere loss. In contrast, outside of subtelomeres, the distribution and orientation of SiRTAs appears random. Since truncating the chromosome at most SiRTAs would be lethal, this observation argues against selection for these sequences as sites of telomere addition per se. We find, however, that sequences predicted to function as SiRTAs are significantly more prevalent across the genome than expected by chance. Sequences identified by the algorithm bind the telomeric protein Cdc13, raising the possibility that association of Cdc13 with single-stranded regions generated during the response to DNA damage may facilitate DNA repair more generally.

 

Fluorescent nucleobase surrogates capable of Watson–Crick hydrogen bonding are essential probes of nucleic acid structure and dynamics, but their limited brightness and short absorption and emission wavelengths have rendered them unsuitable for single-molecule detection. Aiming to improve on these properties, we designed a new tricyclic pyrimidine nucleoside analogue with a push–pull conjugated system and synthesized it in seven sequential steps. The resulting C -linked 8-(diethylamino)benzo[ b ][1,8]naphthyridin-2(1 H )-one nucleoside, which we name ABN, exhibits ε 442 = 20 000 M −1 cm −1 and Φ em,540 = 0.39 in water, increasing to Φ em = 0.50–0.53 when base paired with adenine in duplex DNA oligonucleotides. Single-molecule fluorescence measurements of ABN using both one-photon and two-photon excitation demonstrate its excellent photostability and indicate that the nucleoside is present to > 95% in a bright state with count rates of at least 15 kHz per molecule. This new fluorescent nucleobase analogue, which, in duplex DNA, is the brightest and most red-shifted known, is the first to offer robust and accessible single-molecule fluorescence detection capabilities.