Homologous Recombination and Repair Functions Required for Mutagenicity during Yeast Meiosis

Abstract

Several meiotic events reshape the genome prior to its transfer (via gametes) to the next generation. The occurrence of new meiotic mutations is tightly linked to homologous recombination (HR) and firmly depends on Spo11-induced DNA breaks. To gain insight into the molecular mechanisms governing mutagenicity during meiosis, we examined the timing of mutation and recombination events in cells deficient in various DNA HR-repair genes, which represent distinct functions along the meiotic recombination process. Despite sequence similarities and overlapping activities of the two DNA translocases, Rad54 and Tid1, we observed essential differences in their roles in meiotic mutation occurrence: in the absence of Rad54, meiotic mutagenicity was elevated 8-fold compared to the wild type (WT), while in the tid1Δ mutant, there were few meiotic mutations, nine percent compared to the WT. We propose that the presence of Rad54 channels recombinational repair to a less mutagenic pathway, whereas repair assisted by Tid1 is more mutagenic. A 3.5-fold increase in mutation level was observed in dmc1∆ cells, suggesting that single-stranded DNA (ssDNA) may be a potential source for mutagenicity during meiosis. Taken together, we suggest that the introduction of de novo mutations also contributes to the diversification role of meiotic recombination. These rare meiotic mutations revise genomic sequences and may contribute to long-term evolutionary changes.

Keywords: DNA double-strand breaks (DSBs); DNA repair; homologous recombination; meiosis; mutations.

Figure 1

Kinetics of viability, recombination, and mutation events in WT, dmc1Δ, hed1∆, and double-mutant hed1Δdmc1Δ strains during meiosis. Aliquots of meiotic cells were taken at the times indicated and assayed for (A) cell viability (%) on YEPD plates, (B) recombination at HIS4 on −His plates, and (C) mutation at CAN1 on Can −Ade plates. WT (n = 19, in blue), dmc1∆ (n = 8, in red), hed1∆ (n = 9, in green), and hed1∆dmc1∆ (n = 9, in purple). n denotes the number of independent experiments for each strain. Recombination rate—recombinations per 100 meiotic events. Mutation rate—mutations per million meiotic events. The number of colonies appearing on selection plates from time zero (either on −His or on Can −Ade plates) was always subtracted from the numbers obtained at later time points. Therefore, the net meiotic contribution at the beginning of the experiment is zero.

Figure 2

Kinetics of viability, recombination, and mutation events in WT, rad54Δ, tid1∆, and mus81Δ strains during meiosis. Aliquots of meiotic cells were taken at the times indicated, returned to vegetative growth, and assayed for (A) cell viability (%) on YEPD plates, (B) recombination at HIS4 on −His plates, and (C) mutation at CAN1 on Can −Ade plates. WT (n = 19, in blue), tid1∆ (n = 7, in red), rad54∆ (n = 7, in green), and mus81∆ (n = 6, in purple). In C, the Y axis is split so that the lower half of the plot covers 0 to 1.5 and the upper half of the plot covers 2 to 20 regarding CAN1 mutation rates ×10⁻⁶. n denotes the number of independent experiments for each strain. Recombination rate—recombinations per 100 meiotic events. Mutation rate—mutations per million meiotic events. The number of colonies appearing on selection plates from time zero (either on −His or on Can −Ade plates) was always subtracted from the numbers obtained at later time points. Therefore, the net meiotic contribution at the beginning of the experiment is zero.

Figure 3

Viability, recombination, and mutations obtained at 8 h in WT and in rad54∆ cells upon treatment with the PP1 inhibitor. Aliquots of meiotic cells were taken at 8 h following transfer to SPM, treated with the PP1 inhibitor (dotted bars) or without PP1 (solid bars). (A) Cell viability (%) on YEPD plates. (B) Recombination at HIS4 on −His plates. (C) Mutation at CAN1 on Can −Ade plates. WT (blue); rad54∆ (green).