SITE-SPECIFIC PAUSING OF DEOXYRIBONUCLEIC-ACID SYNTHESIS CATALYZED BY 4 FORMS OF ESCHERICHIA-COLI DNA POLYMERASE-III
SITE-SPECIFIC PAUSING OF DEOXYRIBONUCLEIC-ACID SYNTHESIS CATALYZED BY 4 FORMS OF ESCHERICHIA-COLI DNA POLYMERASE-III
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DOI：
10.1021/bi00291a018
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发表时间：
1983-01-01
期刊：
影响因子：
2.9
通讯作者：
BAMBARA, RA
中科院分区：
文献类型：
--
作者：
LADUCA, RJ;FAY, PJ;BAMBARA, RA
fd噬菌体DNA模板的位点，其终止由4种形式的E. coli DNA聚合酶III的单核苷酸分辨率。通过在高分辨率聚丙烯酰胺凝胶电泳上比较由DNA聚合酶III形式产生的产物与使用双脱氧核苷酸测序方法从相同3“-末端产生的产物来获得结果。每种形式的DNA聚合酶III产生不同长度的产物，终止于有限数量的优选合成终止位点。添加辅助亚基的DNA聚合酶III的核心形式的酶有一个独特的功能上的引物延伸和特异性的聚合酶暂停的影响。大多数位点（65%）可以与通过局部氢键相互作用产生的模板中潜在二级结构的位置相关。聚合酶暂停到邻近发夹茎的位点的接近度与酶的大小有关，因为较小的核心形式的DNA聚合酶III通常在比较大的全酶更接近这些结构的基础的位点处暂停。终止位点的出现明显受亚精胺或大肠杆菌的影响。大肠杆菌单链DNA结合蛋白。此外，观察到暂停位点的核苷酸组成特异性。
Sites of an fd phage DNA template which terminate synthesis catalyzed by each of 4 forms of E. coli DNA polymerase III were identified at single nucleotide resolution. Results were obtained by comparing the products made by forms of DNA polymerase III with products generated from the same 3''-terminus by using the dideoxynucleotide sequencing method, on high-resolution polyacrylamide gel electrophoresis. Each form of DNA polymerase III generates products of distinct lengths ending at a limited number of preferred sites of synthesis termination. The addition of auxiliary subunits to the DNA polymerase III core form of the enzyme has a distinct functional effect on primer elongation and specificity of polymerase pausing. Most sites (65%) can be correlated to positions of potential secondary structure in the template arising via local H-bonding interactions. The proximity of polymerase pausing to sites adjacent to hairpin stems was related to the size of the enzyme since the smaller core form of DNA polymerase III generally paused at sites which were closer to the base of these structures than the larger holoenzyme. The occurrence of termination sites is markedly affected by the inclusion of spermidine or E. coli single-stranded DNA binding protein in the reaction mixtures. Additionally, a nucleotide composition specificity of pause sites is observed.