Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen.
Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen.
复制标题
酿酒酵母核提取物中细胞周期特异性质粒 DNA 复制：复制蛋白 A 和增殖细胞核抗原的调节。
DOI：
10.1021/bi0120289
复制
发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Biswas,SubhasisB
中科院分区：
文献类型：
--
作者：
Mitkova,AtanaskaV;Biswas,EstherE;Biswas,SubhasisB
酵母细胞核提取物中质粒DNA的体外复制与体内复制相似，具有S期特异性。我们在这里报告了一个重建的体外系统与部分纯化的复制蛋白，纯化的复制蛋白A（RPA），和重组增殖细胞核抗原（PCNA）。从S-期，G1-期，和非同步化的酵母细胞的核提取物进行分级磷酸纤维素色谱法。富含复制蛋白的蛋白质组分（聚合酶组分），包括DNA聚合酶（α、δ等），分离到了一个不能在体外复制超螺旋质粒DNA的细胞。然而，当纯化的酵母RPA和重组PCNA一起加入到从S期同步化细胞获得的聚合酶组分中时，体外质粒DNA复制恢复。在体外质粒DNA复制与聚合酶馏分从非同步化和G1期细胞不能重建后，除了纯化的RPA和PCNA。从细胞周期各个时期分离的RPA和PCNA在相同程度上补充了S期聚合酶池。重建系统与S-期聚合酶池，补充与RPA和PCNA-含有馏分或纯化RPA和重组PCNA一起，能够产生复制中间体（大小范围从50到1500 bp）与观察到的S-期核提取物相似。这里提出的结果表明，RPA和PCNA是细胞周期的独立性，在他们的能力，刺激体外质粒DNA复制，而复制因子的聚合酶馏分是严格的S期依赖。
Plasmid DNA replication in nuclear extracts ofSaccharomyces cerevisiaein vitro has been shown to be S-phase specific, similar to that observed in vivo. We report here a reconstituted in vitro system with partially purified replication proteins, purified replication protein A (RPA), and recombinant proliferating cell nuclear antigen (PCNA). Nuclear extracts from S-phase, G1-phase, and unsynchronized yeast cells were fractionated by phosphocellulose chromatography. Protein fraction (polymerase fraction) enriched with replication proteins, including DNA polymerases (α, δ, etc.), was isolated, which was not capable of in vitro replication of supercoiled plasmid DNA. However, when purified yeast RPA and recombinant PCNA together were added to the polymerase fraction obtained from S-phase synchronized cells, in vitro plasmid DNA replication was restored. In vitro plasmid DNA replication with polymerase fractions from unsynchronized and G1-phase cells could not be reconstituted upon addition of purified RPA and PCNA. RPA and PCNA isolated from various phases of the cell cycle complemented the S-phase polymerase pool to the same extent. Reconstituted systems with the S-phase polymerase pool, complemented with either the RPA- and PCNA-containing fraction or purified RPA and recombinant PCNA together, were able to produce replication intermediates (ranging in size from 50 to 1500 bp) similar to that observed with the S-phase nuclear extract. Results presented here demonstrate that both RPA and PCNA are cell cycle-independent in their ability to stimulate in vitro plasmid DNA replication, whereas replication factors in the polymerase fractions are strictly S-phase dependent.