Graves T, Dante M, Eisenhour L, Christianson T W
Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, USA.
Nucleic Acids Res. 1998 Mar 1;26(5):1309-16. doi: 10.1093/nar/26.5.1309.
The active origins of DNA replication for yeast (Saccharomyces cerevisiae) mitochondrial DNA share 280 conserved base pairs and have a promoter. Since intact replication intermediates retain their initiating ribonucleotide triphosphate, we used guanylyltransferase to in vitro cap the replication intermediates present in restriction enzyme-cut DNA from an ori-5 hypersuppressive petite. Restriction mapping and RNA sequencing of these labeled intermediates showed that each DNA strand is primed at a single discrete nucleotide, that one primer starts at the promoter and that the other primer starts 34 nt away, outside the conserved region. Deoxyribonuclease digestion of the capped fragments left resistant RNA primers, which enabled identification of zones of transition from RNA to DNA synthesis. Some of the results contradict the prevailing model for priming at the yeast mitochondrial origins.
酵母（酿酒酵母）线粒体DNA复制的活性起始位点共有280个保守碱基对，并拥有一个启动子。由于完整的复制中间体保留了其起始核糖核苷酸三磷酸，我们使用鸟苷酸转移酶在体外对来自ori - 5超抑制性小菌落的经限制性内切酶切割的DNA中存在的复制中间体进行加帽。对这些标记中间体的限制性图谱分析和RNA测序表明，每条DNA链在单个离散核苷酸处起始引发，一个引物从启动子处开始，另一个引物在保守区域之外34个核苷酸处开始。对加帽片段进行脱氧核糖核酸酶消化后留下抗性RNA引物，这使得能够识别从RNA合成到DNA合成的转变区域。一些结果与酵母线粒体起源处引发的主流模型相矛盾。
