Yoo S K, Ito J
Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson 85724.
J Mol Biol. 1991 Apr 20;218(4):779-89. doi: 10.1016/0022-2836(91)90266-9.
In vitro studies have demonstrated that linear duplex, protein-free DNA molecules containing an inverted terminal repeat (ITR) sequence of the PRD1 genome at one end can undergo replication by a protein-primed mechanism. No DNA replication was observed when the ITR sequence was deleted or was not exposed at the terminus of the template DNA. We have determined the minimal origin of replication by analyzing the template activity of various deletion derivatives. Our results showed that the terminal 20 base-pairs of ITR are required for efficient in vitro DNA replication. We have found that, within the minimal replication origin region, there are complementary sequences. A site-specific mutagenesis analysis showed that most of the point mutations in the complementary sequences markedly reduced the template activity. The analyses of the results obtained with synthetic oligonucleotides have revealed that the specificity of the replication origin is strand specific and even on a single-stranded template a particular DNA sequence including a 3'-terminal C residue is required for the initiation of PRD1 DNA replication in vitro.
体外研究表明，一端含有PRD1基因组反向末端重复（ITR）序列的线性双链无蛋白质DNA分子可通过蛋白质引发机制进行复制。当ITR序列缺失或未暴露于模板DNA末端时，未观察到DNA复制。我们通过分析各种缺失衍生物的模板活性确定了最小复制起点。我们的结果表明，ITR的末端20个碱基对是高效体外DNA复制所必需的。我们发现，在最小复制起点区域内存在互补序列。位点特异性诱变分析表明，互补序列中的大多数点突变显著降低了模板活性。对合成寡核苷酸所得结果的分析表明，复制起点的特异性是链特异性的，即使在单链模板上，体外PRD1 DNA复制起始也需要包含3'末端C残基的特定DNA序列。
