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Molecular and Cellular Biology, January 2002, p. 614-625, Vol. 22, No. 2
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.2.614-625.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Chromatin Assembly Factor I Mutants Defective for PCNA Binding Require Asf1/Hir Proteins for Silencing

Denise C. Krawitz, Tamar Kama, and Paul D. Kaufman*

Lawrence Berkeley National Laboratory and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720

Received 30 May 2001/ Returned for modification 29 June 2001/ Accepted 8 October 2001

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.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, 351 Donner Laboratory, University of California, Berkeley, CA 94720. Phone: (510) 486-5846. Fax: (510) 486-6488. E-mail: pdkaufman{at}lbl.gov.

Molecular and Cellular Biology, January 2002, p. 614-625, Vol. 22, No. 2
0022-538X/01/$04.00+0     DOI: 10.1128/MCB.22.2.614-625.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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