Analysis of covalent complexes formed between calf thymus DNA topoisomerase and single-stranded DNA.
Analysis of covalent complexes formed between calf thymus DNA topoisomerase and single-stranded DNA.
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小牛胸腺 DNA 拓扑异构酶和单链 DNA 之间形成的共价复合物的分析。
DOI：
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发表时间：
1980
期刊：
影响因子：
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通讯作者：
H. Vosberg
中科院分区：
文献类型：
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作者：
B. Prell;H. Vosberg
DNA拓扑异构酶（或缺口闭合酶）在DNA中引入瞬时旋转。研究了小牛胸腺拓扑异构酶与单链fd DNA的反应。这种酶的切口反应伴随着酶与DNA的强烈附着。我们报告了DNA和拓扑异构酶之间的中间复合物形成的条件，并描述了它们的一些性质。为了进行分析，使用了一种过滤器结合试验，该试验基于dna -蛋白质结合物在Whatman玻璃纤维过滤器上的吸附[见Coombs和Pearson (1978) Proc. Natl Acad. Sci.]。《美国科学》第75卷第5期[j]。过滤器上dna拓扑异构酶复合物的出现率是测定中酶浓度的函数。如果酶分子与DNA分子的比例约为50，则发现络合的DNA分子约为9 × 107 fd /s。拓扑异构酶和DNA之间的结合在100 mM KOH或100 mM HCl中稳定，并且抵抗4 M盐酸胍，1 M磷酸钾（pH 6.8）或苯酚的处理。这些结果表明DNA和酶之间存在共价键。此外，小牛胸腺酶附着在缺口处的3 '端。5 '端去磷酸化。poly（dG）和poly（dG）·poly（dC）选择性地抑制单链fd DNA的过滤结合和超螺旋PM2 DNA的松弛，而其他多脱氧核苷酸则不具有抑制作用。这表明该酶更倾向于结合和/或切割DNA中dG或富含dG的簇。
DNA topoisomerases (or nicking-closing enzymes) introduce transient swivels into DNA. We studied the reaction of the calf thymus topoisomerase with single-stranded fd DNA. The nicking reaction of this enzyme is accompanied by strong attachment of the enzyme to DNA. We report conditions under which the intermediate complexes between DNA and topoisomerase are formed and describe some of their properties. For the analyses a filter-binding assay was used which is based on the adsorption of DNA-protein associates to Whatman glass-fiber filters [see Coombs and Pearson (1978) Proc. Natl Acad. Sci. U.S.A. 75, 5291–5295]. The rate of appearance of DNA-topoisomerase complexes on the filter is a function of the enzyme concentration in the assay. The equivalent of about 9 × 107 fd DNA molecules/s are found complexed, if the ratio of enzyme molecules to DNA molecules is about 50. The bond between topoisomerase and DNA is stable in 100 mM KOH or 100 mM HCl and resists treatment with 4 M guanidinium hydrochloride, 1 M potassium phosphate (pH 6.8) or phenol. These results indicate a covalent bond between DNA and the enzyme. Furthermore, the calf thymus enzyme attaches to the 3′ terminus at the nick. The 5′ terminus is dephosphorylated. Filter binding of single-stranded fd DNA as well as relaxation of superhelical PM2 DNA is selectively inhibited by poly(dG) and poly(dG) · poly(dC), but not by other polydeoxynucleotides. This suggests a preference of the enzyme for binding and/or cleaving at dG or dG-rich clusters in the DNA.