RECONSTRUCTION OF BACTERIOPHAGE-T4 DNA-REPLICATION APPARATUS FROM PURIFIED COMPONENTS - ROLLING CIRCLE REPLICATION FOLLOWING DENOVO CHAIN INITIATION ON A SINGLE-STRANDED CIRCULAR DNA TEMPLATE
RECONSTRUCTION OF BACTERIOPHAGE-T4 DNA-REPLICATION APPARATUS FROM PURIFIED COMPONENTS - ROLLING CIRCLE REPLICATION FOLLOWING DENOVO CHAIN INITIATION ON A SINGLE-STRANDED CIRCULAR DNA TEMPLATE
复制标题
DOI：
10.1073/pnas.72.12.4800
复制
发表时间：
1975-01-01
影响因子：
11.1
通讯作者：
ALBERTS, BM
中科院分区：
文献类型：
--
作者：
MORRIS, CF;SINHA, NK;ALBERTS, BM
T4噬菌体基因41、43、45、44和62的蛋白质产物已经用一种测定法纯化到接近同质，该测定法测量它们在被适当的突变噬菌体感染的大肠杆菌细胞的粗裂解物中对DNA合成的刺激。当所有这些蛋白质和T4基因32蛋白质在脱氧核糖核苷和核糖核苷三磷酸存在下孵育时，在单链和双链DNA模板上都发生广泛的DNA合成。对这种体外系统的分析揭示了归因于体内DNA复制的大部分特征：（1）只有当存在核糖核苷三磷酸时，才能在单链DNA模板上发现从头DNA链起始（正如复制叉的“滞后”链上冈崎片段的RNA引发所预期的那样）。(2)以单链环状DNA为模板，合成可以持续许多时间。广泛合成后的产物类似于在电子显微镜中观察到的滚动圈，其中不连续的“滞后”链合成产生长的、无分支的双链尾部。事实上，所有六个突变鉴定的T4复制基因的产品都需要这些合成表明存在一个大的多酶复合物，构成的T4复制装置。
The protein products of T4 bacteriophage genes 41, 43, 45, 44, and 62 have been purified to near homogeneity using an assay which measures their stimulation of DNA synthesis in a crude lysate of Escherichia coli cells in fected by an appropriate mutant phage. When all of these proteins and T4 gene 32 protein are incubated in the presence of deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single and double-stranded DNA templates. Analysis of this in vitro system reveals most of the features attributed to in vivo DNA replication: (1) De novo DNA chain initiation is found on a single-stranded DNA template only if ribonucleoside triphosphates are present (as expected for RNA priming of Okazaki pieces on the "lagging" strand of a replication fork). (2) With single-stranded circular DNA as template, synthesis continues for many doublings. The products after extensive synthesis resemble a rolling circle as visualized in the electron microscope, with discontinuous "lagging" strand synthesis generating a long, unbranched double-stranded tail. The fact that all six mutationally identified T4 replication gene products are required for these syntheses suggests the existence of a large multienzyme complex, constituting the T4 replication apparatus.