Recombination between divergent sequences leads to cell death in a mismatch-repair-independent manner

Abstract

Homologous recombination is an important DNA repair mechanism in vegetative cells. During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences, thus creating a gene-conversion event. Gene conversion of a functional member of a gene family, which uses an inactive member (such as a pseudogene) as a template, might have deleterious consequences. It is therefore important for the cell to prevent recombination between divergent sequences. We have studied the repair of a double-strand break by recombination in a haploid yeast strain carrying 99% identical alleles located on different chromosomes. The fate of the broken chromosome was followed in the whole cell population without imposing selective constraints. Our results show that all the cells were able to repair the broken chromosome by gene conversion. During the repair, the cells arrest in the cell cycle with a "dumbbell" configuration characteristic of G2/M-arrested cells. Surprisingly, although all the cells repaired the broken chromosome, 60% of them were unable to resume growth and to form colonies after the repair was completed. The low level of cell recovery was due to the 1% divergence between the alleles, but was not dependent on the function of the mismatch-repair system. Cell death, however, could be prevented by the presence of an alternative source of perfect homology located on a different chromosome.

Homology Search and Choice of Homologous Partner during Mitotic Recombination

“…It is important to note that consistent with our previous results (16), mitotic gene conversion with both LYS2 and ura3 sequences can take place in vegetative cells in the absence of a functional MMR system. This is different from what the current models for recombination predict and from what is observed in MMR Ϫ cells undergoing meiosis, in which gene conversion is greatly reduced (4,8,14,19,39).…”

“…The second copy, located on another chromosome, carries a similar site containing a single-base-pair mutation that prevents recognition by the endonuclease (ura3-HOcs-inc) (43). In addition, the two ura3 alleles differ at two restriction sites, located to the right (BamHI) and to the left (EcoRI) of the HOcs-inc insertion; these polymorphisms are used to monitor the transfer of genetic information between the chromosomes (15,16). In these strains the HO gene is under the transcriptional control of the GAL1 promoter.…”

“…It is possible to monitor the kinetics of recombination in real time, by extracting DNA from aliquots taken at intervals and subjecting them to Southern blot analysis. This analysis has shown that a DSB is produced in essentially every cell of the population and is repaired efficiently by a gene conversion event that transfers genetic information from chromosome V to chromosome II (15,16).…”

“…(ii) Although deletion of the MMR genes influences the choice between the competing partners, the overall efficiency of repair remained unchanged; 100% of the population still is able to repair the break by gene conversion. Thus, mitotic gene conversion can take place without an active MMR system (16,30). (iii) In several systems in which crossing over can be monitored, mutations in the MMR genes lead to an increase in crossing-over levels (4,8,14,35).…”

Molecular Dissection of Mitotic Recombination in the Yeast Saccharomyces cerevisiae

“…A simple explanation for the coupling and directionality is that a short stretch of the invading DNA is degraded (e.g., by the editing functions of the DNA polymerase) before engaging in DNA synthesis. We favor this model because it has previously been shown that gene conversion in our system is not affected by mutations in the mismatch repair genes (15), whereas mutations in the editing domain of polymerase delta do affect gene conversion (Y. Aylon and M. Kupiec, unpublished data; see also reference 37).…”