Trans-Lesion DNA Polymerases May Be Involved in Yeast Meiosis

Abstract

Trans-lesion DNA polymerases (TLSPs) enable bypass of DNA lesions during replication and are also induced under stress conditions. Being only weakly dependent on their template during replication, TLSPs introduce mutations into DNA. The low processivity of these enzymes ensures that they fall off their template after a few bases are synthesized and are then replaced by the more accurate replicative polymerase. We find that the three TLSPs of budding yeast Saccharomyces cerevisiae Rev1, PolZeta (Rev3 and Rev7), and Rad30 are induced during meiosis at a time when DNA double-strand breaks (DSBs) are formed and homologous chromosomes recombine. Strains deleted for one or any combination of the three TLSPs undergo normal meiosis. However, in the triple-deletion mutant, there is a reduction in both allelic and ectopic recombination. We suggest that trans-lesion polymerases are involved in the processing of meiotic double-strand breaks that lead to mutations. In support of this notion, we report significant yeast two-hybrid (Y2H) associations in meiosis-arrested cells between the TLSPs and DSB proteins Rev1-Spo11, Rev1-Mei4, and Rev7-Rec114, as well as between Rev1 and Rad30 We suggest that the involvement of TLSPs in processing of meiotic DSBs could be responsible for the considerably higher frequency of mutations reported during meiosis compared with that found in mitotically dividing cells, and therefore may contribute to faster evolutionary divergence than previously assumed.

Keywords: DNA repair; DSB processing; meiosis; recombination; trans-lesion synthesis polymerases.

Figure 1

Expression profiles of TLSP genes during yeast meiosis. (A) Levels of RNA of four TLSP genes are shown, based on RNA hybridized to yeast ORF microarrays (Friedlander et al. 2006). Intensity is relative to average abundance levels of the given mRNA. (B) Western blot analysis of TLSP proteins during meiosis. Cells expressing epitope-tagged TLSPs were collected for Western blot analysis at the indicated times after transfer to sporulation conditions. Cdc5 and Ime2 expression peaks mark prophase I and meiotic S phase, respectively (see also Figure 2E). β-Actin was used as a load control.

Figure 2

TLSP protein up-regulation correlates with prophase I events in meiosis. Aliquots of meiotic SK1 cells (strain DAO178) were taken at the indicated times and assayed: (A) Rad30p expression by Western blot analysis; Aco1 was used as a loading control. (B) His⁺ recombinants. Cells were plated on –His medium to produce colonies resulting from homologous recombination at HIS4. (C) Appropriate dilutions were plated on YPD medium, and colonies were assayed for ploidy by mating to mating-type testers (100 colonies per time point). (D) Meiotic cultures were assayed by Western blotting for levels of Rev1 and Dmc1 (strain DAO137) and Tid1 (strain DAO180) proteins. Western blotting time course analysis of these strains was carried out as described in Materials and Methods. The apparent shift in Rev1 band at 6–7 hr is probably the result of distortion of the gel, as it was not seen in four other meiosis time course experiments. (E) Schematic timeline of the major meiotic events during sporulation in strains of SK1 genetic background (see also Székvolgyi and Nicolas 2010).

Figure 3

Sporulation, homologous (allelic) recombination, and ectopic gene conversion in strains homozygous for TLSP gene deletions. (A) Sporulation of cultures of isogenic strains deleted for one, two, or all three TLSP genes and their ancestral WT strain (no TLSP deletions). For each strain, 200 cells were examined at 48 hr, using phase-contrast microscopy. No statistically significant differences were found among sporulation frequencies of different strains (chi-square test). (B) Kinetics during meiosis of allelic recombination at HIS4 and ectopic gene conversion of a point mutation in URA3 in the WT strain. (C) Maximal allelic recombination values at HIS4 for the seven TLSP-deletion strains and their ancestral WT, obtained at 10 or 12 hr in sporulation. Each value is the mean of n independent experiments (n = 8, n = 4, and so forth), and the bars denote SEs. (D) Maximal ectopic gene conversion values of a point mutation at URA3 (ura3-T360G), obtained as described in Figure 3C and in Materials and Methods. Columns in C and D represent the same strains, and results were obtained from the same experiments.

Figure 4

Statistically significant Y2H interactions between TLSPs and meiotic DSB proteins. Shown for each case is the level of β-gal units obtained from a diploid strain carrying the two fusion protein plasmids (left column [gray]) and the two corresponding controls (white): one from a strain carrying one plasmid with the Gal4AD fusion protein and an “empty” plasmid (middle column) and the other, a control, with the plasmid harboring the LexA-BD fusion protein and the other “empty” plasmid (right column). All values are means ± SE obtained from four independent experiments. (A) Values obtained from mitotically dividing cells. (B) Values obtained from meiosis-arrested cells.