Discontinuous synthesis of both strands at the growing fork during polyoma DNA replication in vitro
Discontinuous synthesis of both strands at the growing fork during polyoma DNA replication in vitro
复制标题
多瘤 DNA 体外复制期间生长叉处两条链的不连续合成
作者：
M. Närkhammar;R. Eliasson;G. Magnusson
在不连续的多瘤DNA复制中，冈崎片段的合成是由RNA引发的。在从感染细胞分离的细胞核中合成病毒DNA的过程中，40%的新生短DNA片段具有前导链的极性，理论上，这可以通过连续机制合成。为了排除前导链片段是由新生DNA降解产生的可能性，对它们进行了进一步的表征。复制叉只向一个方向传递的基因组片段的DNA片段被链分离。两条链上的片段大小相似，这表明其中一条链没有被特异性降解。然而，最重要的是，两条链的大部分冈崎片段在其5'端与RNA相连。为了鉴定，RNA在5‘端用[β -32P]GTP标记，在内部用[3H]CTP、[3H]GTP和[3H]UTP标记，或在3’端用相邻[32P]dTMP残基转移的32P标记。所有三种标记表明，来自两条链的DNA片段与RNA引物连接的比例相等。
In discontinuous polyoma DNA replication, the synthesis of Okazaki fragments is primed by RNA. During viral DNA synthesis in nuclei isolated from infected cells, 40% of the nascent short DNA fragments had the polarity of the leading strand which, in theory, could have been synthesized by a continuous mechanism. To rule out that the leading strand fragments were generated by degradation of nascent DNA, they were further characterized. DNA fragments from a segment of the genome which replication forks pass in only one direction were strand separated. The sizes of the fragments from both strands were similar, suggesting that one strand was not specifically degraded. Most important, however, the majority of the Okazaki fragments of both strands were linked to RNA at their 5' ends. For identification, the RNA was labeled at the 5' ends by [beta-32P]GTP, internally by [3H]CTP, [3H]GTP, and [3H]UTP, or at the 3' ends by 32P transfer from adjacent [32P]dTMP residues. All three kinds of labeling indicated that an equal proportion of DNA fragments from the two strands was linked to RNA primers.