Wittmeyer J, Formosa T
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132, USA.
Mol Cell Biol. 1997 Jul;17(7):4178-90. doi: 10.1128/MCB.17.7.4178.
We have used DNA polymerase alpha affinity chromatography to identify factors involved in eukaryotic DNA replication in the yeast Saccharomyces cerevisiae. Two proteins that bound to the catalytic subunit of DNA polymerase alpha (Pol1 protein) are encoded by the essential genes CDC68/SPT16 and POB3. The binding of both proteins was enhanced when extracts lacking a previously characterized polymerase binding protein, Ctf4, were used. This finding suggests that Cdc68 and Pob3 may compete with Ctf4 for binding to Pol1. Pol1 and Pob3 were coimmunoprecipitated from whole-cell extracts with antiserum directed against Cdc68, and Pol1 was immunoprecipitated from whole-cell extracts with antiserum directed against the amino terminus of Pob3, suggesting that these proteins may form a complex in vivo. CDC68 also interacted genetically with POL1 and CTF4 mutations; the maximum permissive temperature of double mutants was lower than for any single mutant. Overexpression of Cdc68 in a pol1 mutant strain dramatically decreased cell viability, consistent with the formation or modulation of an essential complex by these proteins in vivo. A mutation in CDC68/SPT16 had previously been shown to cause pleiotropic effects on the regulation of transcription (J. A. Prendergrast et al., Genetics 124:81-90, 1990; E. A. Malone et al., Mol. Cell. Biol. 11:5710-5717, 1991; A. Rowley et al., Mol. Cell. Biol. 11:5718-5726, 1991), with a spectrum of phenotypes similar to those caused by mutations in the genes encoding histone proteins H2A and H2B (Malone et al., Mol. Cell. Biol. 11:5710-5717, 1991). We show that at the nonpermissive temperature, cdc68-1 mutants arrest as unbudded cells with a 1C DNA content, consistent with a possible role for Cdc68 in the prereplicative stage of the cell cycle. The cdc68-1 mutation caused elevated rates of chromosome fragment loss, a phenotype characteristic of genes whose native products are required for normal DNA metabolism. However, this mutation did not affect the rate of loss or recombination for two intact chromosomes, nor did it affect the retention of a low-copy-number plasmid. The previously uncharacterized Pob3 sequence has significant amino acid sequence similarity with an HMG1-like protein from vertebrates. Based on these results and because Cdc68 has been implicated as a regulator of chromatin structure, we postulate that polymerase alpha may interact with these proteins to gain access to its template or to origins of replication in vivo.
我们利用DNA聚合酶α亲和层析法来鉴定参与酿酒酵母真核DNA复制的因子。与DNA聚合酶α催化亚基（Pol1蛋白）结合的两种蛋白质由必需基因CDC68/SPT16和POB3编码。当使用缺乏先前已鉴定的聚合酶结合蛋白Ctf4的提取物时，这两种蛋白的结合增强。这一发现表明，Cdc68和Pob3可能与Ctf4竞争结合Pol1。用针对Cdc68的抗血清从全细胞提取物中共免疫沉淀出Pol1和Pob3，并用针对Pob3氨基末端的抗血清从全细胞提取物中免疫沉淀出Pol1，这表明这些蛋白质可能在体内形成复合物。CDC68在遗传上也与POL1和CTF4突变相互作用；双突变体的最高允许温度低于任何单突变体。在pol1突变菌株中过表达Cdc68会显著降低细胞活力，这与这些蛋白质在体内形成或调节必需复合物一致。先前已表明，CDC68/SPT16中的突变会对转录调控产生多效性影响（J. A. Prendergrast等人，《遗传学》124:81 - 90，1990；E. A. Malone等人，《分子细胞生物学》11:5710 - 5717，1991；A. Rowley等人，《分子细胞生物学》11:5718 - 5726，1991），其一系列表型类似于由编码组蛋白H2A和H2B的基因突变所导致的表型（Malone等人，《分子细胞生物学》11:5710 - 5717，1991）。我们发现，在非允许温度下，cdc68 - 1突变体停滞为具有1C DNA含量的未出芽细胞，这与Cdc68在细胞周期复制前阶段可能发挥的作用一致。cdc68 - 1突变导致染色体片段丢失率升高，这是正常DNA代谢所需天然产物的基因所具有的表型特征。然而，该突变并不影响两条完整染色体的丢失或重组率，也不影响低拷贝数质粒的保留。先前未鉴定的Pob3序列与脊椎动物的一种HMG1样蛋白具有显著的氨基酸序列相似性。基于这些结果，并且由于Cdc68已被认为是染色质结构的调节因子，我们推测聚合酶α可能与这些蛋白质相互作用，以便在体内接触其模板或复制起点。
