Nielsen B L, Rajasekhar V K, Tewari K K
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Plant Mol Biol. 1991 Jun;16(6):1019-34. doi: 10.1007/BF00016074.
A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.
从豌豆叶绿体中分离出一种DNA引发酶活性，并对其在复制中的作用进行了研究。通过从DEAE - 纤维素柱上进行线性盐梯度洗脱，将DNA引发酶活性与大部分叶绿体RNA聚合酶活性分离，然后通过肝素 - 琼脂糖柱分别纯化这两种酶活性。引发酶活性不受叶绿体RNA聚合酶的特异性抑制剂tagetitoxin或针对纯化的豌豆叶绿体RNA聚合酶制备的多克隆抗体的抑制，而RNA聚合酶活性则被tagetitoxin或多克隆抗体完全抑制。DNA引发酶活性能够在包括聚（dT）、聚（dC）、M13mp19和M13mp19 + 2.1在内的单链模板上引发DNA复制，M13mp19 + 2.1包含富含AT的豌豆叶绿体复制起点。RNA聚合酶组分在使用这些单链DNA模板中的任何一种进行体外复制反应时，都无法支持3H - TTP的掺入。甘油梯度分析表明，豌豆叶绿体DNA引发酶（115 - 120 kDa）与豌豆叶绿体DNA聚合酶（90 kDa）分离，但比叶绿体RNA聚合酶小得多。由于在大小、模板特异性、对抑制剂的敏感性和洗脱特性方面存在这些差异，很明显豌豆叶绿体DNA引发酶是一种与RNA聚合酶不同的酶。使用DNA引发酶组分的体外复制活性需要所有四种rNTP才能达到最佳活性。叶绿体DNA引发酶能够在任何单链M13模板上引发DNA复制活性，但对M13mp19 + 2.1表现出强烈偏好。使用M13mp19 + 2.1合成的引物对DNase I有抗性，大小范围从4到约60个核苷酸。
