Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA.
Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA.
复制标题
基因 6 核酸外切酶在噬菌体 T7 DNA 复制和包装中的作用。
DOI：
10.1016/s0022-2836(05)80347-x
复制
发表时间：
1990
影响因子：
5.6
通讯作者：
Son,M
中科院分区：
文献类型：
--
作者：
Serwer,P;Watson,RH;Son,M
当噬菌体T7基因6外切核酸酶从T7感染的细胞中遗传性地去除时，观察到细胞内T7 DNA的降解。本研究采用高速区带离心，然后进行脉冲场琼脂糖凝胶电泳或限制性内切酶分析，观察到以下结果。(1)大多数细胞内DNA的降解需要T7基因3核酸内切酶的存在，并且不依赖于DNA包装;当基因3和基因6产物都不存在时，快速沉降的分支DNA积累。(2)相对少量的降解需要包装，并且发生在多联体中基因组之间的接合处和细胞内DNA的左端附近; DNA包装仅部分被阻断，并且基因组的端对端连接在不存在gene 6核酸外切酶的情况下不被阻断。(3)在不存在基因6核酸外切酶的情况下产生的片段是线性的并且不进一步降解;片段的前体是非线性的。(4)产生这些片段的一些但不是大多数裂解选择性地发生在两个已知的DNA复制起点附近。基于这些观察，得出结论，在T7基因6外切核酸酶不存在下发生的大多数降解是由分支处的切割引起的。提出了以下假设：大多数，可能是所有的，由去除基因6核酸外切酶诱导的额外分支是由DNA合成的RNA引物位点处的链置换DNA合成引起的;由DNA复制的多次启动产生的RNA引物，在野生型T7感染期间被基因6核酸外切酶的RNA酶H活性去除。在不存在gene 6核酸外切酶的情况下对基因组连接的观察和其他观察表明，多联体化所需的单链末端重复序列是由DNA复制产生的。观察到的选择性缩短的左端表明，gene 6核酸外切酶是需要形成的大部分，可能是所有的，成熟的左端。
When bacteriophage T7 gene6exonuclease is genetically removed from T7-infected cells, degradation of intracellular T7 DNA is observed. By use of rate zonal centrifugation, followed by either pulsed-field agarose gel electrophoresis or restriction endonuclease analysis, in the present study, the following observations were made. (1) Most degradation of intracellular DNA requires the presence of T7 gene3endonuclease and is independent of DNA packaging; rapidly sedimenting, branched DNA accumulates when both the gene3and gene6products are absent. (2) A comparatively small amount of degradation requires packaging and occurs at both the joint between genomes in a concatemer and near the left end of intracellular DNA; DNA packaging is only partially blocked and end-to-end joining of genomes is not blocked in the absence of gene6exonuclease. (3) Fragments produced in the absence of gene6exonuclease are linear and do not further degrade; precursors of the fragments are non-linear. (4) Some, but not most, of the cleavages that produce these fragments occur selectively near two known origins of DNA replication. On the basis of these observations, the conclusion is drawn that most degradation that occurs in the absence of T7 gene6exonuclease is caused by cleavage at branches. The following hypothesis is presented: most, possibly all, of the extra branching induced by removal of gene6exonuclease is caused by strand displacement DNA synthesis at the site of RNA primers of DNA synthesis; the RNA primers, produced by multiple initiations of DNA replication, are removed by the RNase H activity of gene6exonuclease during a wild-type T7 infection. Observation of joining of genomes in the absence of gene6exonuclease and additional observations indicate that single-stranded terminal repeats required for concatamerization are produced by DNA replication. The observed selective shortening of the left end indicates that gene6exonuclease is required for formation of most, possibly all, mature left ends.