Telomere Loops and Homologous Recombination-Dependent Telomeric Circles in a Kluyveromyces lactis Telomere Mutant Strain

Abstract

The Kluyveromyces lactis ter1-16T strain contains mutant telomeres that are poorly bound by Rap1, resulting in a telomere-uncapping phenotype and significant elongation of the telomeric DNA. The elongated telomeres of ter1-16T allowed the isolation and examination of native yeast telomeric DNA by electron microscopy. In the telomeric DNA isolated from ter1-16T, looped molecules were observed with the physical characteristics of telomere loops (t-loops) previously described in mammalian and plant cells. ter1-16T cells were also found to contain free circular telomeric DNA molecules (t-circles) ranging up to the size of an entire telomere. When the ter1-16T uncapping phenotype was repressed by overexpression of RAP1 or recombination was inhibited by deletion of rad52, the isolated telomeric DNA contained significantly fewer t-loops and t-circles. These results suggest that disruption of Rap1 results in elevated recombination at telomeres, leading to increased strand invasion of the 3′ overhang within t-loop junctions and resolution of the t-loop junctions into free t-circles.

ACKNOWLEDGMENTS

Roger R. Reddel (CMRI, Westmead, Australia) is thanked for the use of his laboratory's apparatus and reagents to complete some of the 2D gels shown here.

This work was supported by grants from the National Institutes of Health to J.D.G. (GM31819 and ES 013773) and to M.J.M. (GM61645). C.G.-V. was supported through a training grant from the National Institutes of Health (5T32GMOO&10330) and S.A.C. though a National Institutes of Heath postdoctoral training grant (GM077900-01).