Published online 1 September 2005
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Reduction of nucleosome assembly during new DNA synthesis impairs both major pathways of double-strand break repair
Department of Chemistry and Biochemistry, Texas State University San Marcos, TX 78666, USA 1Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH Research Triangle Park, NC 27709, USA
*To whom correspondence should be addressed. Tel: +1 512 245 8594; Fax: +1 512 245 2374; Email: LL18{at}txstate.edu
Received August 5, 2005. Accepted August 15, 2005.
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 
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.
Present address: G. Karthikeyan, Office of Science and Technology, Duke University, Durham, NC 27710, USA
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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