Noskov V, Maki S, Kawasaki Y, Leem S H, Ono B, Araki H, Pavlov Y, Sugino A
Department of Genetics, Sankt-Petersburg University, Sankt-Petersburg, Russia.
Nucleic Acids Res. 1994 May 11;22(9):1527-35. doi: 10.1093/nar/22.9.1527.
Replication Factor C (RF-C) of Saccharomyces cerevisiae is a complex that consists of several different polypeptides ranging from 120- to 37 kDa (Yoder and Burgers, 1991; Fien and Stillman, 1992), similar to human RF-C. We have isolated a gene, RFC2, that appears to be a component of the yeast RF-C. The RFC2 gene is located on chromosome X of S. cerevisiae and is essential for cell growth. Disruption of the RFC2 gene led to a dumbbell-shaped terminal morphology, common to mutants having a defect in chromosomal DNA replication. The steady-state levels of RFC2 mRNA fluctuated less during the cell cycle than other genes involved in DNA replication. Nucleotide sequence of the gene revealed an open reading frame corresponding to a polypeptide with a calculated Mr of 39,716 and a high degree of amino acid sequence homology to the 37-kDa subunit of human RF-C. Polyclonal antibodies against bacterially expressed Rfc2 protein specifically reduced RF-C activity in the RF-C-dependent reaction catalyzed by yeast DNA polymerase III. Furthermore, the Rfc2 protein was copurified with RF-C activity throughout RF-C purification. These results strongly suggest that the RFC2 gene product is a component of yeast RF-C. The bacterially expressed Rfc2 protein preferentially bound to primed single-strand DNA and weakly to ATP.
酿酒酵母的复制因子C（RF-C）是一种由几种不同的多肽组成的复合体，分子量范围从120 kDa到37 kDa（约德和伯格斯，1991年；菲恩和斯蒂尔曼，1992年），与人类RF-C相似。我们分离出了一个基因RFC2，它似乎是酵母RF-C的一个组成部分。RFC2基因位于酿酒酵母的X染色体上，对细胞生长至关重要。RFC2基因的破坏导致了哑铃状的末端形态，这在染色体DNA复制有缺陷的突变体中很常见。RFC2 mRNA的稳态水平在细胞周期中的波动比其他参与DNA复制的基因要小。该基因的核苷酸序列揭示了一个开放阅读框，对应于一个计算分子量为39716的多肽，并且与人类RF-C的37 kDa亚基有高度的氨基酸序列同源性。针对细菌表达的Rfc2蛋白的多克隆抗体在酵母DNA聚合酶III催化的依赖RF-C的反应中特异性地降低了RF-C的活性。此外，在整个RF-C纯化过程中，Rfc2蛋白与RF-C活性一起被共纯化。这些结果强烈表明RFC2基因产物是酵母RF-C的一个组成部分。细菌表达的Rfc2蛋白优先结合引发的单链DNA，与ATP的结合较弱。
