Influence of DNA Sequence Identity on Efficiency of Targeted Gene Replacement

Abstract

We have developed a system for analyzing recombination between a DNA fragment released in the nucleus from a single-copy plasmid and a genomic target in order to determine the influence of DNA sequence mismatches on the frequency of gene replacement in Saccharomyces cerevisiae. Mismatching was shown to be a potent barrier to efficient gene replacement, but its effect was considerably ameliorated by the presence of DNA sequences that are identical to the genomic target at one end of a chimeric DNA fragment. Disruption of the mismatch repair gene MSH2 greatly reduces but does not eliminate the barrier to recombination between mismatched DNA fragment and genomic target sequences, indicating that the inhibition of gene replacement with mismatched sequences is at least partially under the control of mismatch repair. We also found that mismatched sequences inhibited recombination between a DNA fragment and the genome only when they were close to the edge of the fragment. Together these data indicate that while mismatches can destabilize the relationship between a DNA fragment and a genomic target sequence, they will only do so if they are likely to be in the heteroduplex formed between the recombining molecules.DNA sequences engineered in vitro can be readily introduced into the genome of Saccharomyces cerevisiae cells by homologous recombination, facilitating the creation of duplications, insertions, and deletions of anything from single genes to large chromosomal fragments (33,38). In contrast, early experiments with mammalian cells showed that recombinant DNA molecules are most often inserted randomly into the genome by a mechanism that does not require extensive identity between the DNA fragment and the genomic sequences (32,40,47). More recently, it was shown that the efficiency of homologous gene replacement in mammalian cells can be greatly enhanced relative to random integration by using DNA sequences from a source that is isogenic to the recipient cells, suggesting that the presence of mismatches between the DNA fragment and the genome strongly inhibits homologous recombination (10, 45).DNA sequence mismatching presents a considerable barrier to homologous recombination in a wide variety of systems (5, 9, 10, 14, 21, 31, 34-36, 41, 45, 49, 50). Several laboratories have observed that defects in the mismatch repair machinery in bacterial species greatly lower the barrier against recombination between mismatched sequences (12,18,25,29,36,54). Other investigators have shown that mutations in mismatch repair genes in yeast (9, 34) and mammal (11) cells similarly reduce the inhibitory effect of mismatches, indicating that this genetic mechanism is evolutionarily conserved. It has been suggested that nonidentical sequences are prevented from recombining because the mispairing that occurs when heteroduplex DNA is formed is recognized by the mismatch repair machinery, after which the heteroduplex is unwound (9) or multiply nicked (27). The mismatch repair machinery in yeast corrects mismatches in the heteroduplex fo...

Novel Function of Rad27 (FEN-1) in Restricting Short-Sequence Recombination

“…We used the insertion of DNA fragments into the genome to study the effect of DNA sequence length on homologous recombination in wild-type and mutant yeast. This approach was chosen because DNA fragment length can be readily changed and because DNA fragment insertion is similar to the repair of broken chromosomes by homologous recombination (30,36,38). In this assay, homozygous wild-type and rad27 mutant diploids were transformed with three different DNA fragments consisting of a URA3 marker flanked by varying lengths of HIS3 sequence.…”

Allele-specific gene targeting in Candida albicans results from heterology between alleles The GenBank accession numbers for the sequences reported in this paper are AF247189 and AF247190 for PHR1-1 and PHR1-2, respectively.

“…Recombination between like alleles was a homologous event while recombination between the diverged alleles was a homeologous event. It is well established that sequence heterology interferes with recombination in prokaryotic and eukaryotic species (Chen & Jinks-Robertson, 1998 ;Elliott et al, 1998 ;Nassif & Engels, 1993 ;Stambuk & Radman, 1998) and this includes targeted gene replacements (Leung et al, 1997 ;Negritto et al, 1996). In S. cerevisiae a single nucleotide difference within a 350 bp region can reduce recombination fourfold (Datta et al, 1997).…”

RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae

“…The his3-Δ3′-HOcs allele was constructed and introduced into the HIS3 locus of chromosome XV as follows: pUC-HIS, which carries the 1.8 kb wild-type HIS3 sequence cloned into the BamHI site of pUC18 [38], was used to create pLAY498 by removing the KpnI site from the polylinker. Subsequently, a 127 bp KpnI/XhoI fragment from pLAY97 [39] carrying the HOcs was used to replace a 238 bp KpnI/XhoI fragment of pLAY498 containing the final 41 bp of the HIS3 coding sequence, creating his3-Δ3′-HOcs on pLAY500. The construct was excised from pLAY500 by digestion with BamHI and inserted into the BamHI site of the integrating plasmid YIp356R [40] to create pLAY504.…”

“…The rad1 mutant displayed the greatest defect of all the single mutants with translocation frequencies that were 600-fold lower than wild-type with the 300 bp substrates and 93-fold lower with the 60 bp substrates. Since the structure-specific nuclease Rad1/Rad10, and the mismatch repair heterodimer Msh2/Msh3 have been shown to work together to process recombination intermediates, particularly during SSA [39,58,59,60], we also tested an msh2 mutant and observed a substantial defect in this strain as well, with frequencies that were similar to those of the rad1 mutant. A similar 100-fold reduction in the frequency of translocation was also observed with the 300 bp substrates in the DSB repair mutants rad52, mre11, and rad59.…”