Recombinational telomere elongation in Kluyveromyces lactis

Abstract

There are at least two pathways of telomere maintenance in yeast and mammalian cells, one being telomerase-mediated and the other being dependent on homologous recombination. Yeast cells normally depend on telomerase to maintain their telomeres. We propose that recombination-dependent rolling circle replication around a circular template may be a mechanism by which telomeres are elongated in a single step. This could lead to the formation of post-senescence survivors among cells that lack telomerase activity. We have found that DNA circles of 1.6 kb, composed of telomeric repeats and a marker gene can be used as templates to form tandem arrays at telomeres. The process is partially dependent on RAD52, a gene involved in homologous recombination. The elongated sequence derived from a DNA circle may spread to most or all telomeres in a cell by gene conversion events that initiate at subtelomeric or telomeric positions. We have also analyzed some features of telomere elongation templated by DNA circles to gain better understanding of the process. A host strain that has elevated recombination rates at telomeres can efficiently utilize a circular telomeric template to form and spread tandem arrays, but a host strain with mostly fused chromosomes is not efficient at either process. A circular template is far more efficient at forming telomeric tandem arrays than an equivalent linear insert. Subtelomeric sequences present in the circular template may affect the initial alignment between the template and the host telomeric end, leading to the occurrence of telomeric deletions in the arrays that form. Either strand of a URA3-telomere circle can be utilized to form telomeric tandem arrays although the presence of 3' G-rich tails at chromosome ends suggests that the C-rich strand would be preferentially utilized. Similarly, either strand of 100 nt telomeric circles can promote the formation of telomeric tandem arrays. We have observed putative telomeric circles of this size in a telomerase-template mutant that has very long telomeres. This suggests that uncapped telomeres can promote the formation of very small circles.