Miranda E I, Garrido-Guerrero E, Garcia-Carranca A, Gariglio P
Department of Genetics and Molecular Biology, Centro de Investigacion y de Estudios Avanzados IPN, Mexico.
Nucleic Acids Res. 1992 Feb 25;20(4):903-7. doi: 10.1093/nar/20.4.903.
Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin. Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes. Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes. When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated. Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates. Protein analysis of the pelleted material revealed several proteins of viral and cellular origin. Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes. Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots. These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein. The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin.
猴病毒40（SV40）DNA复制是研究真核细胞DNA复制的一个有用模型，因为它仅编码一种复制蛋白，并且其基因组具有与细胞染色质无法区分的核蛋白结构（“微型染色体”）。感染后期，SV40复制性DNA分子约占病毒微型染色体总数的5%。由于噬菌体T4的基因32蛋白（P32）与单链DNA相互作用，并且预计SV40复制复合物在复制叉处含有单链区域，因此我们询问P32是否可用于分离正在复制的SV40微型染色体。当用P32和抗P32抗体依次处理来自SV40感染细胞的核提取物时，脉冲标记的微型染色体被选择性免疫沉淀。琼脂糖凝胶电泳分析证实免疫沉淀的物质对应于SV40复制中间体。对沉淀物质的蛋白质分析揭示了几种病毒和细胞来源的蛋白质。其中，T抗原和组蛋白被发现与至少其他三种细胞来源的蛋白质结合在复制复合物上。此外，抗P32抗体能够在蛋白质印迹中检测到三种分子量约为70、32和13 kDa的细胞蛋白。这些蛋白质可能对应于作为真核多亚基单链DNA结合蛋白一部分所发现的那些蛋白质。因此，使用P32和抗P32抗体可以将正在复制的SV40微型染色体与成熟的微型染色体分离，并且可以构成一种富集和研究复制活性染色质的新方法。
