Belanger K G, Mirzayan C, Kreuzer H E, Alberts B M, Kreuzer K N
Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Nucleic Acids Res. 1996 Jun 1;24(11):2166-75. doi: 10.1093/nar/24.11.2166.
The rolling circle DNA replication structures generated by the in vitro phage T4 replication system were analyzed using two-dimensional agarose gels. Replication structures were generated in the presence or absence of T4 primase (gp61), permitting the analysis of replication forks with either duplex or single-stranded tails. A characteristic arc shape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that anneals to restriction sites in the single-stranded tail. After calibrating the gel system with this well-studied rolling circle replication reaction, we then analyzed the in vivo replication directed by a T4 replication origin cloned within a plasmid. DNA samples were generated from infections with either wild-type or primase-deletion mutant phage. The only replicative arc that could be detected in the wild-type sample corresponded to duplex Y forms, consistent with very efficient lagging strand synthesis. Surprisingly, we obtained evidence for both duplex and single-stranded DNA tails in the samples from the primase-deficient infection. We conclude that a relatively inefficient mechanism primes lagging strand DNA synthesis in vivo when gp61 is absent.
利用二维琼脂糖凝胶对体外噬菌体T4复制系统产生的滚环DNA复制结构进行了分析。在有或没有T4引发酶（gp61）的情况下产生复制结构，从而能够分析具有双链或单链尾巴的复制叉。当具有单链尾巴的复制叉在一种寡核苷酸的帮助下被一种限制酶切割时，会呈现出一种特征性的弧形，该寡核苷酸可与单链尾巴中的限制位点退火。在用这个经过充分研究的滚环复制反应校准凝胶系统后，我们接着分析了由克隆在质粒中的T4复制起点指导的体内复制。DNA样本来自野生型或引发酶缺失突变体噬菌体感染。在野生型样本中能够检测到的唯一复制弧对应于双链Y形，这与非常高效的后随链合成一致。令人惊讶的是，我们在引发酶缺陷感染的样本中获得了双链和单链DNA尾巴的证据。我们得出结论，当不存在gp61时，一种相对低效的机制在体内引发后随链DNA合成。
