Tsutsui K, Watanabe S, Katagiri M, Oda T
Nucleic Acids Res. 1983 Jul 25;11(14):4793-807. doi: 10.1093/nar/11.14.4793.
Protein species interacting with newly replicated DNA were analyzed using a photo cross-linking technique. Nascent DNA was labeled in vitro with [alpha-32P]dCTP and BrdUTP in SV40-infected CV-1 cells made permeable with saponin. The labeled cells were then irradiated with UV light (254 nm) and were treated extensively with DNase I. Proteins with radioactive DNA tags were separated by SDS-PAGE and visualized by autoradiography. Among 10-15 proteins which were cross-linked, the proteins with apparent molecular weights of 16.5 K, 44 K, 82 K and those in the 94-140 K region appeared to be associated with newly replicated SV40 DNA. A pulse-chase experiment showed that the 82 K and 94-140 K proteins interacted with new DNA in a relatively localized region close to the replication fork. The 44 K protein was identified as the major viral capsid protein, VP1, using antiserum to SV40 capsid proteins. It was suggested that VP1 binds to nascent DNA shortly after DNA synthesis and migrates into chromatin maturation regions.
利用光交联技术分析了与新复制DNA相互作用的蛋白质种类。在经皂角苷处理变得通透的感染了SV40的CV-1细胞中，用[α-32P]dCTP和BrdUTP对新生DNA进行体外标记。然后用紫外线（254nm）照射标记的细胞，并用DNase I进行大量处理。带有放射性DNA标签的蛋白质通过SDS-PAGE分离，并通过放射自显影进行可视化。在10 - 15种发生交联的蛋白质中，表观分子量为16.5K、44K、82K以及94 - 140K区域的蛋白质似乎与新复制的SV40 DNA相关。脉冲追踪实验表明，82K和94 - 140K的蛋白质在靠近复制叉的相对局部区域与新DNA相互作用。使用针对SV40衣壳蛋白的抗血清，将44K的蛋白质鉴定为主要的病毒衣壳蛋白VP1。有人提出，VP1在DNA合成后不久就与新生DNA结合，并迁移到染色质成熟区域。
