Recombination at Long Mutant Telomeres Produces Tiny Single- and Double-Stranded Telomeric Circles

Abstract

Recombinational telomere elongation (RTE) known as alternate lengthening of telomeres is the mechanism of telomere maintenance in up to 5 to 10% of human cancers. The telomeres of yeast mutants lacking telomerase can also be maintained by recombination. Previously, we proposed the roll-and-spread model to explain this elongation in the yeast Kluveromyces lactis. This model suggests that a very small ( approximately 100-bp) circular molecule of telomeric DNA is copied by a rolling circle event to generate a single long telomere. The sequence of this primary elongated telomere is then spread by recombination to all remaining telomeres. Here we show by two-dimensional gel analysis and electron microscopy that small circles of single- and double-stranded telomeric DNA are commonly made by recombination in a K. lactis mutant with long telomeres. These circles were found to be especially abundant between 100 and 400 bp (or nucleotides). Interestingly, the single-stranded circles consist of only the G-rich telomeric strand sequence. To our knowledge this is the first report of single-stranded telomeric circles as a product of telomere dysfunction. We propose that the small telomeric circles form through the resolution of an intratelomeric strand invasion which resembles a t-loop. Our data reported here demonstrate that K. lactis can, in at least some circumstances, make telomeric circles of the very small sizes predicted by the roll-and-spread model. The very small circles seen here are both predicted products of telomere rapid deletion, a process observed in both human and yeast cells, and predicted templates for roll-and-spread RTE.