title: Reduction of nucleosome assembly during new DNA synthesis impairs both major pathways of double-strand break repair.
abstract: Assembly of new chromatin during S phase requires the histone chaperone complexes CAF-1 (Cac2p, Msi1p and Rlf2p) and RCAF (Asf1p plus acetylated histones H3 and H4). Cells lacking CAF-1 and RCAF are hypersensitive to DNA-damaging agents, such as methyl methanesulfonate and camptothecin, suggesting a possible defect in double-strand break (DSB) repair. Assays developed to quantitate repair of defined, cohesive-ended break structures revealed that DSB-induced plasmid:chromosome recombination was reduced approximately 10-fold in RCAF/CAF-1 double mutants. Recombination defects were similar with both chromosomal and plasmid targets in vivo, suggesting that inhibitory chromatin structures were not involved. Consistent with these observations, ionizing radiation-induced loss of heterozygosity was abolished in the mutants. Nonhomologous end-joining (NHEJ) repair proficiency and accuracy were intermediate between wild-type levels and those of NHEJ-deficient yku70 and rad50 mutants. The defects in NHEJ, but not homologous recombination, could be rescued by deletion of HMR-a1, a component of the a1/alpha2 transcriptional repressor complex. The findings are consistent with the observation that silent mating loci are partially derepressed. These results demonstrate that defective assembly of nucleosomes during new DNA synthesis compromises each of the known pathways of DSB repair and that the effects can be indirect consequences of changes in silenced chromatin structure.
authors.affiliation: Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX 78666, USA. LL18@txstate.edu
authors.forename: L Kevin
authors.initials: LK
authors.lastname: Lewis
authors.forename: G
authors.initials: G
authors.lastname: Karthikeyan
authors.forename: Jared
authors.initials: J
authors.lastname: Cassiano
authors.forename: Michael A
authors.initials: MA
authors.lastname: Resnick
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000235:genetics; D006657:Histones / Q000235:genetics; D019656:Loss of Heterozygosity; D018832:Molecular Chaperones; D009153:Mutagens / Q000633:toxicity; D009154:Mutation; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism*; D011839:Radiation, Ionizing; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: C109731:ASF1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D006657:Histones; D018832:Molecular Chaperones; D009153:Mutagens; D009707:Nucleosomes; D029701:Saccharomyces cerevisiae Proteins