Telomerase Deficiency Affects the Formation of Chromosomal Translocations by Homologous Recombination in Saccharomyces cerevisiae

Abstract

Telomerase is a ribonucleoprotein complex required for the replication and protection of telomeric DNA in eukaryotes. Cells lacking telomerase undergo a progressive loss of telomeric DNA that results in loss of viability and a concomitant increase in genome instability. We have used budding yeast to investigate the relationship between telomerase deficiency and the generation of chromosomal translocations, a common characteristic of cancer cells. Telomerase deficiency increased the rate of formation of spontaneous translocations by homologous recombination involving telomere proximal sequences during crisis. However, telomerase deficiency also decreased the frequency of translocation formation following multiple HO-endonuclease catalyzed DNA double-strand breaks at telomere proximal or distal sequences before, during and after crisis. This decrease correlated with a sequestration of the central homologous recombination factor, Rad52, to telomeres determined by chromatin immuno-precipitation. This suggests that telomerase deficiency results in the sequestration of Rad52 to telomeres, limiting the capacity of the cell to repair double-strand breaks throughout the genome. Increased spontaneous translocation formation in telomerase-deficient yeast cells undergoing crisis is consistent with the increased incidence of cancer in elderly humans, as the majority of our cells lack telomerase. Decreased translocation formation by recombinational repair of double-strand breaks in telomerase-deficient yeast suggests that the reemergence of telomerase expression observed in many human tumors may further stimulate genome rearrangement. Thus, telomerase may exert a substantial effect on global genome stability, which may bear significantly on the appearance and progression of cancer in humans.

DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

“…Identification of the reciprocal translocation (see Fig. 1A) occurred only in a small subset of His + recombinants (1/14), which is similar to previous reports (55,56) (Table S2). To ascertain whether our assay fulfills the criteria that define MMEJ, we examined the effect of ku70Δ, lig4Δ, and rad52Δ single mutants on the frequency of MMEJ repair.…”

“…Newly dissected mutant spores of rad52::TRP1, pol4::KANMX rad52:: TRP1 pol3-ct rad52::TRP1, and rad1::LEU2 rad52::TRP1 double mutants possessing the interchromosomal MMEJ substrates, which share 14-2-4 bp of MH, were grown in YPD overnight, diluted to an OD 600 of 0.2 in fresh YP-Raffinose, and allowed to grow to an OD 600 of ∼1.0, at which time 45 mL was removed (0 h), pelleted, and immediately subject to the ChIP procedure described previously (56). Galactose was added to the remaining culture to a final concentration of 2% (vol/vol), which induces the expression of HO endonuclease and DSB formation at the his3Δ3′ allele.…”

RAD59 and RAD1 cooperate in translocation formation by single-strand annealing in Saccharomyces cerevisiae

“…1; Meyer and Bailis 2008; Pannunzio et al 2008). DSBs are initiated adjacent to each of the truncated alleles that share either 60 or 311 bp of sequence homology, so as to mimic the approximate size of common repetitive elements.…”

Msh2 Blocks an Alternative Mechanism for Non-Homologous Tail Removal during Single-Strand Annealing in Saccharomyces cerevisiae

“…We previously demonstrated that simultaneous HO endonuclease-catalyzed DSBs adjacent to appropriately oriented 60 or 300 bp repetitive sequences on different chromosomes in wild-type diploid cells results in the appearance of chromosomal translocations in 1 to 10% of the survivors (Figure 1, Table 1) [38], [39]. This process, referred to as T2, utilizes two truncated copies of the HIS3 gene that share 60 or 300 bp homologous segments [38].…”