Investigation of the genetics of Recombinational Telomere Elongation (RTE) and its steps in the milk yeast Kluyveromyces lactis

Abstract

In the absence of telomerase certain cancers achieve immortalization through telomere elongation using Alternative Lengthening of Telomeres pathway, which is dependent on telomeric homologous recombination (HR). The process of recombinational telomere elongation (RTE) has been extensively studied in yeast. Upon deletion of telomerase, yeast cells lose most telomeric repeats and die, but rare survivors emerge with elongated telomeres. Saccharomyces cerevisiae can utilize either Type I or Type II RTE which rely on RAD51-dependent or RAD50/RAD59/SGS1-dependent recombination machinery, respectively. Kluyveromyces lactis telomerase deletion mutants generate Type II survivors only. Rare K. lactis telomerase deletion (ter1-∆) survivors emerge using RTE that is thought to occur via a three-step mechanism called the roll- and-spread model. According to this model, a telomeric circle (t-circle) is formed and utilized as a template for DNA synthesis to produce a long telomere. The sequence from a lengthened telomere is then spread to other short telomeres through break-induced replication events. To study the genetics of RTE in K. lactis, mutants lacking telomerase and one or more recombination genes (RAD50, RAD51, RAD59, and SGS1) were created. Both, the RAD51 and RAD50/RAD59/SGS1 pathways act together in a single Type II survivor formation pathway in K. lactis. RAD51, but not RAD59, was required for telomere copying events through recombination originating specifically in subtelomeric regions. RAD59 is essential in the absence of RAD51 as indicated by the absence of survivors in the ter1-∆ rad51-∆ rad59-∆ triple mutant. Furthermore, we have addressed the genetics of the steps postulated to be involved in the roll-and-spread model. Small t-circles were dependent on RAD50, while large t-circles were diminished in the absence of RAD50 and RAD52 indicating that an alternative pathway of t-circle formation may exist. Our results also demonstrate that RTE in phenotypically similar mutants, but carrying different telomere capping mutations, has different genetic requirements. We also found that RTE can occur in the presence of partially defective telomerase. As such, yeast cells can use recombination to either moderately elongate telomeres via the roll-and-spread mechanism or simply to maintain extremely short telomeres.