The highly processive DNA polymerase of bacteriophage T5 - Role of the unique N and C termini
The highly processive DNA polymerase of bacteriophage T5 - Role of the unique N and C termini
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DOI：
10.1074/jbc.m408428200
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发表时间：
2004-11-26
影响因子：
4.8
通讯作者：
Richardson, CC
中科院分区：
文献类型：
--
作者：
Andraos, N;Tabor, S;Richardson, CC
以前报道过噬菌体T5编码的DNA聚合酶是一种进程性的酶，可以催化广泛的链位移合成。这种酶是从噬菌体感染的细胞中纯化出来的，不需要辅助蛋白来进行这些活性。虽然T5 DNA聚合酶与大肠杆菌DNA聚合酶I和T7 DNA聚合酶具有广泛的序列同源性，但它在N端和C端分别含有130个和71个残基的独特区域。我们克隆了野生型T5 DNA聚合酶的编码基因，并对过量产生的蛋白进行了鉴定。我们还研究了N端和c端缺失对加工能力和链位移合成的影响。缺乏N端30残基的T5 DNA聚合酶与野生型酶相似，但聚合酶活性降低了2倍。C端缺失24个残基导致引物环状DNA上聚合酶活性降低30倍，加工能力显著降低，无法进行链置换合成。C端缺失63个残基导致聚合酶活性降低2万倍。与T5 DNA聚合酶相关的3‘至5’双链DNA外切酶活性在N端被截断的聚合酶中被降低了5倍，而在C端被截断的聚合酶中被刺激了7倍。我们提出了一种模型，其中C端增加了DNA对聚合酶活性位点的亲和力，从而增加了DNA对外切酶活性位点的亲和性并降低了DNA对外切酶活性位点的可及性。
The DNA polymerase encoded by bacteriophage T5 has been reported previously to be processive and to catalyze extensive strand displacement synthesis. The enzyme, purified from phage-infected cells, did not require accessory proteins for these activities. Although T5 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I and T7 DNA polymerase, it contains unique regions of 130 and 71 residues at its N and C termini, respectively. We cloned the gene encoding wild-type T5 DNA polymerase and characterized the overproduced protein. We also examined the effect of N- and C-terminal deletions on processivity and strand displacement synthesis. T5 DNA polymerase lacking its N- terminal 30 residues resembled the wild-type enzyme albeit with a 2-fold reduction in polymerase activity. Deletion of 24 residues at the C terminus resulted in a 30-fold reduction in polymerase activity on primed circular DNA, had dramatically reduced processivity, and was unable to carry out strand displacement synthesis. Deletion of 63 residues at the C terminus resulted in a 20,000-fold reduction in polymerase activity. The 3' to 5' double-stranded DNA exonuclease activity associated with T5 DNA polymerase was reduced by a factor of 5 in the polymerase truncated at the N terminus but was stimulated by a factor of 7 in the polymerase truncated at the C terminus. We propose a model in which the C terminus increases the affinity of the DNA for the polymerase active site, thus increasing processivity and decreasing the accessibility of the DNA to the exonuclease active site.