Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing
Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing
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DOI：
10.1128/mcb.22.2.614-625.2002
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发表时间：
2002-01-01
影响因子：
5.3
通讯作者：
Kaufman, PD
中科院分区：
文献类型：
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作者：
Krawitz, DC;Kama, T;Kaufman, PD
染色质组装因子1（Chromatin assembly factor 1，CAF-I）是一种保守的组蛋白H3/H4沉积复合物。缺乏CAF-I亚基基因（CAC 1至CAC 3）的酿酒酵母突变体显示出减少的异染色质基因沉默。在筛选沉默受损的cac 1等位基因时，我们分离出一个突变，该突变减少了与Cac 3 p亚基的结合，另一个突变则削弱了与DNA复制蛋白PCNA的结合。令人惊讶的是，在Cac 1 p的突变，废除PCNA结合导致非常轻微的端粒沉默缺陷，但造成沉默在很大程度上依赖于Hir蛋白和Asf 1 p，这两者共同构成了一个替代沉默途径。与这些表型相一致，突变型CAF-I复合物缺陷的PCNA结合显示减少核小体组装活性在体外，但刺激Asf 1 p-组蛋白复合物。此外，这些突变的CAF-I复合物显示出降低的偏好沉积组蛋白到新复制的DNA。我们还观察到Asf 1 p和Cac 2 p在体外的弱相互作用，我们假设，这种相互作用的基础这些组蛋白沉积蛋白之间的功能协同作用。
Chromatin assembly factor 1 (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.