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含胸腺嘧啶光二聚体的单链或双链寡脱氧核糖核苷酸对T4内切核酸酶V的亲和力。

Affinity of single- or double-stranded oligodeoxyribonucleotides containing a thymine photodimer for T4 endonuclease V.

作者信息

Inaoka T, Ishida M, Ohtsuka E

机构信息

Faculty of Pharmaceutical Sciences, Hokaido University, Sapporo, Japan.

出版信息

J Biol Chem. 1989 Feb 15;264(5):2609-14.

PMID:2914925
Abstract

A gene for T4 endonuclease V was constructed by joining chemically synthesized oligodeoxyribonucleotides and expressed efficiently in Escherichia coli under the control of the E. coli tryptophan promoter. Overproduced T4 endonuclease V, which can cleave thymine photodimers as well as the corresponding phosphodiester linkage of DNA, was used to investigate the precise mode of the reaction with single- or double-stranded synthetic DNA fragments containing a thymine photodimer. The substrates, three oligodeoxyribonucleotides, d(GCGGTTGGCG) (10-mer), d(CGAAGGTTGGAAGC) (14-mer), and d(CACGAAGGTTGGAAGCAC) (18-mer), were prepared by UV irradiation of the nascent oligonucleotides. These single-stranded oligonucleotides were cleaved by the enzyme with a concentration 100 times higher than that required for the corresponding duplexes. The Km values for the TT duplex (14- and 18-mer) were found to be on the order of 10(-8) M. Dissociation constants for the 14- and 18-mer duplexes were measured by a binding assay on a nitrocellulose filter and found to be 10(-9).

摘要

通过连接化学合成的寡脱氧核糖核苷酸构建了T4核酸内切酶V基因，并在大肠杆菌色氨酸启动子的控制下在大肠杆菌中高效表达。过量产生的T4核酸内切酶V能够切割胸腺嘧啶光二聚体以及DNA相应的磷酸二酯键，用于研究其与含有胸腺嘧啶光二聚体的单链或双链合成DNA片段反应的精确模式。通过对新生寡核苷酸进行紫外线照射制备了三种寡脱氧核糖核苷酸底物，即d(GCGGTTGGCG)（10聚体）、d(CGAAGGTTGGAAGC)（14聚体）和d(CACGAAGGTTGGAAGCAC)（18聚体）。这些单链寡核苷酸被该酶切割时所需的浓度比相应双链体所需浓度高100倍。发现TT双链体（14聚体和18聚体）的Km值约为10^(-8) M。通过在硝酸纤维素滤膜上进行结合测定，测得14聚体和18聚体双链体的解离常数为10^(-9)。

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1
Affinity of single- or double-stranded oligodeoxyribonucleotides containing a thymine photodimer for T4 endonuclease V.含胸腺嘧啶光二聚体的单链或双链寡脱氧核糖核苷酸对T4内切核酸酶V的亲和力。
J Biol Chem. 1989 Feb 15;264(5):2609-14.
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Reaction mechanism of T4 endonuclease V determined by analysis using modified oligonucleotide duplexes.通过使用修饰的寡核苷酸双链体分析确定T4核酸内切酶V的反应机制。
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Endonuclease V from bacteriophage T4 interacts with its substrate in the minor groove.来自噬菌体T4的核酸内切酶V在小沟中与其底物相互作用。
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