Stabilization of yeast artificial chromosome clones in a rad54-3 recombination-deficient host strain

Nucleic Acids Res. 1997 Mar 15;25(6):1248-53. doi: 10.1093/nar/25.6.1248.


The cloning and propagation of large fragments of DNA on yeast artificial chromosomes (YACs) has become a routine and valuable technique in genome analysis. Unfortunately, many YAC clones have been found to undergo rearrangements or deletions during the cloning process. The frequency of transformation-associated alterations and mitotic instability can be reduced in a homologous recombination-deficient yeast host strain such as a rad52 mutant. RAD52 is one member of an epistatic group of genes required for the recombinational repair of double-strand breaks in DNA. rad52 mutants grow more slowly and transform less efficiently than RAD + strains and are therefore not ideal hosts for YAC library construction. We have investigated the ability of both null and temperature-sensitive alleles of RAD54 , another member of the RAD52 epistasis group, to prevent rearrangements of human YAC clones containing tandemly repeated DNA sequences. Our results show that the temperature-sensitive rad54-3 allele blocks mitotic recombination between tandemly repeated DYZ3 satellite sequences and significantly stabilizes a human DYZ5 satellite-containing YAC clone. Yeast carrying the rad54-3 mutation can undergo meiosis, have growth and transformation rates comparable with RAD + strains, and therefore represent improved YAC cloning hosts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromosomes, Artificial, Yeast*
  • DNA Helicases
  • DNA Repair Enzymes
  • DNA, Satellite / genetics
  • Fungal Proteins / genetics*
  • Fungal Proteins / metabolism
  • Gene Library
  • Genotype
  • Humans
  • Mating Factor
  • Meiosis
  • Mitosis
  • Mutagenesis
  • Peptides / genetics
  • Recombination, Genetic*
  • Repetitive Sequences, Nucleic Acid*
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins*
  • Transformation, Genetic


  • DNA, Satellite
  • Fungal Proteins
  • Peptides
  • Saccharomyces cerevisiae Proteins
  • Mating Factor
  • RAD54 protein, S cerevisiae
  • DNA Helicases
  • DNA Repair Enzymes