Bypass of heterology during strand transfer by Saccharomyces cerevisiae Rad51 protein.
During recombination-mediated repair of DNA double-strand breaks, strand transfer proteins must distinguish a homologous repair template from closely related genomic sequences. However, some tolerance by strand transfer proteins for sequence differences is also critical: too much stringency will prevent recombination between different alleles of the same gene, but too much tolerance will lead to illegitimate recombination. We characterized the heterology tolerance of Saccharomyces cerevisiae Rad51 by testing bypass of small heterologous inserts in either the single- or double-stranded substrate of an in vitro strand transfer reaction that models the early steps of homologous recombination. We found that the yeast protein is rather stringent, only tolerating heterologies up to 9 bases long. The efficiency of heterology bypass depends on whether the insert is in the single- or double-stranded substrate, as well as on the location of the insert relative to the end of the double-stranded linear substrate. Rad51 is distinct in that it can catalyze strand transfer in either the 3'-->5' or 5'-->3' direction. We found that bypass of heterology was independent of the polarity of strand transfer, suggesting that the mechanism of 5'-->3' transfer is the same as that of 3'-->5' transfer.
Holmes
V F
VF
Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720-3204, USA.
Benjamin
K R
KR
Crisona
N J
NJ
Cozzarelli
N R
NR
D004247:DNA / Q000235:genetics / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D051135:Rad51 Recombinase; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004254:DNA Nucleotidyltransferases; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase