Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing.
Chromatin assembly factor I (CAF-I) is a conserved histone H3/H4 deposition complex. Saccharomyces cerevisiae mutants lacking CAF-I subunit genes (CAC1 to CAC3) display reduced heterochromatic gene silencing. In a screen for silencing-impaired cac1 alleles, we isolated a mutation that reduced binding to the Cac3p subunit and another that impaired binding to the DNA replication protein PCNA. Surprisingly, mutations in Cac1p that abolished PCNA binding resulted in very minor telomeric silencing defects but caused silencing to be largely dependent on Hir proteins and Asf1p, which together comprise an alternative silencing pathway. Consistent with these phenotypes, mutant CAF-I complexes defective for PCNA binding displayed reduced nucleosome assembly activity in vitro but were stimulated by Asf1p-histone complexes. Furthermore, these mutant CAF-I complexes displayed a reduced preference for depositing histones onto newly replicated DNA. We also observed a weak interaction between Asf1p and Cac2p in vitro, and we hypothesize that this interaction underlies the functional synergy between these histone deposition proteins.
Krawitz
Denise C
DC
Lawrence Berkeley National Laboratory and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.
Kama
Tamar
T
Kaufman
Paul D
PD
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics*; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics; D020868:Gene Silencing; D005800:Genes, Fungal; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D021122:Protein Subunits; D012097:Repressor Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D016615:Telomere / Q000235:genetics
C109731:ASF1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C078808:HIR1 protein, S cerevisiae; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; C533589:RLF2 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins