Masai H, Arai K
Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304.
J Biol Chem. 1988 Oct 15;263(29):15016-23.
The role of the primosome assembly and protein n' recognition site in replication of pBR322 plasmid was examined. The following evidence indicates that the primosome is involved in lagging-strand synthesis of pBR322 plasmid replication in vitro. Early replicative intermediates with newly synthesized leading strand, approximately 1 kilobase pair long, immediately downstream of the replication origin accumulate in products synthesized in extracts from a dnaT strain that lacks primosomal protein i or in wild-type extracts supplemented with anti-protein i antibody. These intermediates are converted efficiently into full-length DNA by addition of purified protein i. Consistent with the previously proposed role of the primosome (Arai, K. and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 69-73), an n' site on the lagging strand, but not on the leading strand, is required for efficient replication of the plasmid in vitro. Plasmids lacking an n' site on the lagging strand replicate only to a limited extent in vitro and early replicative intermediates carrying nascent leading strands are accumulated, although a portion of the intermediates complete replication to yield full-length DNA. The latter reaction is completely inhibited by addition of anti-protein i antibody. Insertion of the n' site of phage phi X174 into pBR322 plasmids lacking lagging-strand n' sites restores the replicative ability of the mutant plasmid comparable to that of the wild-type plasmid. These results indicate that protein i is essential for lagging-strand synthesis of pBR322 plasmid in vitro and that it may play an important role in the priming events as a part of either an n' site-dependent primosome or an n' site-independent, as yet unidentified, priming complex.
研究了引发体组装和蛋白质n'识别位点在pBR322质粒复制中的作用。以下证据表明，引发体参与了体外pBR322质粒复制的滞后链合成。在缺乏引发体蛋白i的dnaT菌株提取物或补充了抗蛋白i抗体的野生型提取物中合成的产物中，积累了具有新合成的前导链的早期复制中间体，该前导链在复制起点下游约1千碱基对长。通过添加纯化的蛋白i，这些中间体可有效地转化为全长DNA。与先前提出的引发体作用一致（荒井，K.和科恩伯格，A.（1981年）美国国家科学院院刊78，69 - 73），滞后链上的n'位点而非前导链上的n'位点是质粒体外高效复制所必需的。缺乏滞后链n'位点的质粒在体外仅有限程度地复制，并且携带新生前导链的早期复制中间体积累，尽管一部分中间体完成复制产生全长DNA。加入抗蛋白i抗体可完全抑制后一种反应。将噬菌体phi X174的n'位点插入缺乏滞后链n'位点的pBR322质粒中，可恢复突变体质粒的复制能力，使其与野生型质粒相当。这些结果表明，蛋白i对于体外pBR322质粒的滞后链合成至关重要，并且它可能作为n'位点依赖性引发体或n'位点非依赖性、尚未鉴定的引发复合物的一部分，在引发事件中发挥重要作用。
