Fine-resolution mapping of spontaneous and double-strand break-induced gene conversion tracts in Saccharomyces cerevisiae reveals reversible mitotic conversion polarity.

Abstract

Spontaneous and double-strand break (DSB)-induced gene conversion was examined in alleles of the Saccharomyces cerevisiae ura3 gene containing nine phenotypically silent markers and an HO nuclease recognition site. Conversions of these alleles, carried on ARS1/CEN4 plasmids, involved interactions with heteroalleles on chromosome V and were stimulated by DSBs created at HO sites. Crossovers that integrate plasmids into chromosomes were not detected since the resultant dicentric chromosomes would be lethal. Converted alleles in shuttle plasmids were easily transferred to Escherichia coli and analyzed for marker conversion, facilitating the characterization of more than 400 independent products from five crosses. This analysis revealed several new features of gene conversions. The average length of DSB-induced conversion tracts was 200 to 300 bp, although about 20%o were very short (less than 53 bp). About 20%o of spontaneous tracts also were also less than 53 bp, but spontaneous tracts were on average about 40%o longer than DSB-induced tracts. Most tracts were continuous, but 3% had discontinuous conversion patterns, indicating that extensive heteroduplex DNA is formed during at least this fraction of events. Mismatches in heteroduplex DNA were repaired in both directions, and repair tracts as short as 44 bp were observed. Surprisingly, most DSB-induced gene conversion tracts were unidirectional and exhibited a reversible polarity that depended on the locations of DSBs and frameshift mutations in recipient and donor alleles.Gene conversion is defined as the nonreciprocal transfer of two strands of information from one DNA duplex to a homologous duplex. Gene conversion has been widely studied in yeasts and other fungi and is evidenced during meiosis by 6:2 aberrant segregation patterns (43). Gene conversion also occurs in mitotic cells, e.g., yeast mating-type interconversion (21). Although gene conversion involves nonreciprocal information transfer, it is often associated with reciprocal exchange of markers flanking the converted region. Such exchanges are explained by models that invoke symmetric cross-strand structures (Holliday junctions [22]) that allow resolution of recombination intermediates in two senses, predicting flanking marker exchange in up to 50% of products (41).An important feature of gene conversion is that close markers often coconvert. This feature indicates that gene conversion involves a region of DNA or tract, and many studies have shown that most conversion tracts are continuous (1,3,7,8,26,61,67). Conversion tract lengths have been measured for both meiotic and mitotic events. A controlled study comparing tract lengths for allelic URA3 genes indicated that mitotic tracts were usually longer than 2 to 4 kbp, whereas meiotic tracts were usually shorter than 2 kbp (26). Average tract lengths were less than 500 bp for mitotic conversion between alleles in plasmids (3) or between nontandem duplications in a chromosome (1, 67). These shorter tracts probably reflect limitations imposed by the len...

High frequency mitotic gene conversion in genetic hybrids of the oomycete Phytophthora sojae

“…Thus the simultaneous conversion of 8127 and 8801 in some derivatives of NC123 is likely to have occurred by independent events, and the conversion tracts in these cases therefore are likely to have been less than 700 bp. In Saccharomyces cerevisiae, mitotic gene conversion tracts ranged from 50 to 400 bp in length (38,39).…”

The Relationship between Homology Length and Crossing Over during the Repair of a Broken Chromosome

“…The ura3-HOcs alleles were created by inserting a 39-bp oligonucleotide (14) at the NcoI site of the 5.6-kb BamHI fragment containing the URA3 gene. 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).…”

Homology Search and Choice of Homologous Partner during Mitotic Recombination

“…Strain OI29 carries at the LYS2 locus on chromosome II a 1.2-kb HindIII fragment containing the ura3-HOcs allele. This allele was created by inserting a 39-bp oligonucleotide at the NcoI site of the URA3 gene (43). The ura3-HOcs-inc allele was similarly created and differs from the ura3-HOcs allele at three positions: it carries a G-to-A change at the HOcs, which prevents the recognition by the HO endonuclease (26), and substitutions to the right and to the left of the HOcs, which introduce BamHI and EcoRI sites, respectively.…”

“…Strain OI76 was constructed by deleting the MMR genes of strain MK181 by one-or two-step transplacement with plasmid pSR395 for MSH2, pRK366 for MSH3, and pRK465 for MSH6 (4,9). Strain MK182 is identical to strain MK181 except that it bears, at the LYS2 locus on chromosome II, a 1.2-kb HindIII fragment containing the ura3-HOcs allele, into which changes were introduced by site-specific mutagenesis at ϳ100-bp intervals (43). Strain OI77 was created by deleting the MSH2, MSH3, and MSH6 genes of strain MK182 as explained above.…”