Abstract
Homologous recombination can result in the transfer of genetic information from one DNA molecule to another (gene conversion). These events are often accompanied by a reciprocal exchange between the interacting molecules (termed "crossing over"). This association suggests that the two types of events could be mechanistically related. We have analyzed the repair, by homologous recombination, of a broken chromosome in yeast. We show that gene conversion can be uncoupled from crossing over when the length of homology of the interacting substrates is below a certain threshold. In addition, a minimal length of homology on each broken chromosomal arm is needed for crossing over. We also show that the coupling between gene conversion and crossing over is affected by the mismatch repair system; mutations in the MSH2 or MSH6 genes cause an increase in the crossing over observed for short alleles. Our results provide a mechanism to explain how chromosomal recombinational repair can take place without altering the stability of the genome.
Highlights
Double-strand breaks (DSBs)1 in the DNA of living organisms occur as a consequence of the natural cell metabolism, or they can be created by exogenous sources such as chemical agents or radiation
Homologous recombination can result in the transfer of genetic information from one DNA molecule to another
The mechanism suggested by the synthesisdependent strand annealing (SDSA) model accounts mainly for gene conversion events, the DSB in the second model leads to the formation of an intermediate, which can be resolved either as a gene conversion event or as a gene conversion accompanied by a crossover [4]
Summary
Strains—All of the yeast strains used in this study are isogenic derivatives of strain OI27 (MATa-inc ura3-HOcs-inc ade3::GALHO ade leu 112 his trp can1-100) [13]. The different alleles used were subclones of this fragment inserted at a HpaI site within LYS2 sequences in the integrative plasmid pOI5 (TRP1 LYS2 URA3). These alleles were integrated into the yeast chromosome II by a two-step replacement method, selecting first for Trpϩ transformants and plating on 5-FOA plates to obtain TrpϪUraϪ derivatives, which have replaced the LYS2 allele on chromosome II by the appropriate lys2::ura3-HOcs allele. All of the strain configurations were checked by Southern blot analysis.
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