Integrity of human YACs during propagation in recombination-deficient yeast strains

Several isogenic strains with defects in recombination/repair genes (RAD1, RAD50, RAD51, RAD52, RAD54, and RAD55) were examined for their ability to p ropagate accurately a variety of linear and circular yeast artificial chrom osomes (YACs) containing human DNA inserts. To assess YAC stability, the hu man DNA inserts were internally marked by an ADE2-pBR-URA3 cassette. Follow ing selection for Ura(-) clones on 5-fluoroorotic acid containing medium, t he following types of YAC deletions were identified: (i) those caused by ho mologous recombination with a telomeric pBR sequence; (ii) internal deletio ns, presumed to occur by recombination between commonly occurring DNA repea ts such as Alu and LINE sequences; and (iii) deletions leading to loss of p art of a YAC arm. rad52 host strains, but not other recombination-deficient strains, decreased the rate of all types of YAC deletions 25- to 400-fold. We have also developed and tested kar1 strains with a conditional RAD52 ge ne that allow transfer of a YAC from any host into a recombination-deficien t background. These strains provide an efficient tool for stabilization of YACs and are useful for allowing additional recombinational modification of YACs. (C) 1999 Academic Press.