Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi, 467-8603, Japan.
Mol Biol Rep. 2012 Apr;39(4):4989-96. doi: 10.1007/s11033-011-1295-z. Epub 2011 Dec 9.
Histone eviction and deposition are critical steps in many nuclear processes. The histone H3/H4 chaperone Asf1p is highly conserved and is involved in DNA replication, DNA repair, and transcription. To identify the factors concerned with anti-silencing function 1 (ASF1), we purified Asf1p-associated factors from the yeast Saccharomyces cerevisiae by a GST pull-down experiment, and mass spectrometry analysis was performed. Several factors are specifically associated with Asf1p, including Vip1p. VIP1 is conserved from yeast to humans and encodes inositol hexakisphoshate and inositol heptakisphosphate kinase. Vip1p interacted with Asf1p as a dimer or in a complex with another protein(s). Deletion of VIP1 did not affect the interaction between Asf1p and other Asf1p-associated factors. An in vitro GST pull-down assay indicated a direct interaction between Asf1p and Vip1p, and the interaction between the two factors in vivo was detected by an immunoprecipitation experiment. Furthermore, genetic experiments revealed that VIP1 disruption increased sensitivity to 6-azauracil (6-AU), but not to DNA-damaging reagents in wild-type and ASF1-deleted strains. It is thought that 6-AU decreases nucleotide levels and reduces transcription elongation. These observations suggest that the association of Asf1p and Vip1p may be implicated in transcription elongation.
组蛋白的逐出和沉积是许多核过程的关键步骤。组蛋白 H3/H4 伴侣蛋白 Asf1p 高度保守，参与 DNA 复制、DNA 修复和转录。为了鉴定与抗沉默功能 1（ASF1）相关的因子，我们通过 GST 下拉实验从酵母酿酒酵母中纯化了与 Asf1p 相关的因子，并进行了质谱分析。几种因子与 Asf1p 特异性相关，包括 Vip1p。VIP1 从酵母到人都保守，编码肌醇六磷酸和肌醇七磷酸激酶。Vip1p 作为二聚体或与另一种蛋白质复合物与 Asf1p 相互作用。VIP1 的缺失并不影响 Asf1p 与其他 Asf1p 相关因子之间的相互作用。体外 GST 下拉实验表明 Asf1p 和 Vip1p 之间存在直接相互作用，体内免疫沉淀实验检测到这两种因子之间的相互作用。此外，遗传实验表明，VIP1 缺失增加了对 6-氮杂尿嘧啶（6-AU）的敏感性，但对野生型和 ASF1 缺失菌株中的 DNA 损伤试剂没有影响。据认为，6-AU 降低核苷酸水平并减少转录延伸。这些观察结果表明，Asf1p 和 Vip1p 的结合可能与转录延伸有关。
