Mutation of tryptophan 128 in T4 endonuclease V does not affect glycosylase or abasic site lyase activity.
Mutation of tryptophan 128 in T4 endonuclease V does not affect glycosylase or abasic site lyase activity.
复制标题
T4 核酸内切酶 V 中色氨酸 128 的突变不会影响糖基酶或脱碱基位点裂解酶活性。
DOI：
10.1021/bi00196a021
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Lloyd,RS
中科院分区：
文献类型：
--
作者：
Latham,KA;Carmical,JR;Lloyd,RS
1994年5月20日收到的修订稿摘要：核酸内切酶V的羧基末端11个氨基酸残基的各种突变，证明了该区域在二聚体特异性结合中的重要性。在先前的一项研究中，用丝氨酸残基取代色氨酸128会导致蛋白质的碱性裂解酶活性降低，而不会伴随DNA糖基酶活性的降低[Nakabppu，Y.等人(1982)J.Biol]。化学。257,2556-2562]，为了评估128位色氨酸的重要性，通过定点突变构建了6个突变体，包括W128Y、W128V、W128I、W128G、W128S和W128T。经鉴定，这6个突变体可以弥补紫外光照射的大肠杆菌细胞(recA~，uvrA~)的修复缺陷，达到与野生型核酸内切酶V相当的水平。用紫外光照射的质粒DNA和含有位点特异的环丁烷嘧啶二聚体或碱性位点的寡核苷酸对突变蛋白的活性进行了鉴定。在所有的情况下，6个突变体都表现出与野生型内切酶V相似的糖基酶和碱性裂解酶活性，表明Trp-128对于二聚体特异性结合或催化不是关键的。T4噬菌体的DENV试剂编码内切酶V，这是一个很好的DNA修复酶，在紫外线诱导的1-嘧啶二聚体位置切割DNA。这种16 kDa的酶通过盐依赖的一维扩散沿双链DNA定位嘧啶二聚体(Gruskin&Lloyd，1986,1988；Dowd&Lloyd，1989a，b)。当与其底物结合时，该酶裂解二聚体中5‘嘧啶的碱基和糖之间的A-糖基键(Seawell等人，1980；McMillan等人，1981)。两个二聚化的嘧啶之间的磷酸二酯键随后被破坏，留下3‘a，/3-不饱和醛和5’磷酸(Weiss&Grossman，1987)。核酸内切酶V还可以通过a/3消除机制将DNA3‘切割到碱性(AP)位点(Manoharan等人，1988；Mazumder和Gerlt，)。该实验室最近的研究表明，核酸内切酶V的一个Eu末端参与催化(SchRock&Lloyd，1991,1993)，反应通过涉及该酶N-末端的亚氨基中间体进行[Dodson等人，1993；在Latham和Lloyd(1994)中综述]。
Revised Manuscript Received May 20, 1994s abstract: Mutation of various residues within the carboxy-terminal 11 amino acids of endonuclease V, an enzyme made up of 138 amino acids that initiates the repair of cyclobutane pyrimidine dimers in DNA, has demonstrated the importance of this region in dimer-specific binding. In a previous study, substitution of a serine residue for tryptophan 128 resulted in a protein with decreased abasic site lyase activity without a concomitant decrease in DNA glycosylase activity [Nakabeppu, Y., et al.(1982) J. Biol. Chem. 257, 2556-2562], To assess the importance of the tryptophan at position 128, six mutants were constructed by site-directed mutagenesis, including W128Y, W128V, W128I, W128G, W128S, and W128T. Upon characterization, these six mutants were found qualitatively to complement the repair deficiencyof ultraviolet (UV) light irradiatedEscherichia coli cells (recA~, uvrA~) to levels comparable to that of wild-type endonuclease V. The activities of themutant proteins were characterized using UV-irradiated plasmid DNA and oligonucleotidescontaining either a site-specific cyclobutane pyrimidine dimer or an abasic site. In all cases, the six mutants displayed glycosylase and abasic site lyase activities comparable to those of wild-type endonuclease V, indicating that Trp-128 is not crucial for dimer-specific binding or catalysis.The denV gent of bacteriophage T4 encodes endonuclease V, a well-characterized DNA repair enzyme that cleaves DNA at the site of UV-induced1 pyrimidine dimers. This 16-kDa enzyme locates pyrimidine dimers by a salt-dependent one-dimensional diffusion along double-stranded DNA (Gruskin & Lloyd, 1986, 1988; Dowd & Lloyd, 1989a, b). Upon binding its substrate, the enzyme cleaves the A-glycosyl bond between the base and sugar of the 5'pyrimidine within the dimer (Seawell et al., 1980; McMillan et al., 1981). The phos-phodiester bond between the two dimerized pyrimidines is subsequently broken, leaving a 3'a,/3-unsaturated aldehyde and a 5'phosphate (Weiss & Grossman, 1987). Endonuclease V can also cleave DNA 3'to an abasic (AP) site via a/3-elimination mechanism (Manoharan et al., 1988; Mazumder & Gerlt, 1989). Recent studies in thislaboratory have demonstrated that the aNEU-terminus of endonuclease V is involved in catalysis (Schrock & Lloyd, 1991, 1993) and that the reaction proceeds through an imino intermediate involving the N-terminus of the enzyme [Dodson et al., 1993; reviewed in Latham and Lloyd (1994)].
登录
查看更多内容
T4 核酸内切酶 V 的 X 射线结构：一种嘧啶二聚体特异性切除修复酶。
影响因子：
56.9
作者：
Kosuke Morikawa;Osamu Matsumoto;Michiko Tsujimoto;K. Katayanagi;M. Ariyoshi;Takefumi Doi;Morio Ikehara;Tetsuya Inaoka;Eiko Ohtsuka
通讯作者：
Eiko Ohtsuka
影响因子：
2.9
作者：
John-Stephen Taylor;Daniel S. Garrett;I. Brockie;Daniel L. Svoboda;J. Telser
通讯作者：
John-Stephen Taylor;Daniel S. Garrett;I. Brockie;Daniel L. Svoboda;J. Telser
噬菌体 T4 中 denV 基因的鉴定、物理图谱位置和序列。
影响因子：
14.9
作者：
Kristoffer Valerie;E. Henderson;J. K. Deriel
通讯作者：
J. K. Deriel
紫外线照射大肠杆菌内质粒 DNA 修复的分子分析。
DOI：
10.1016/s0021-9258(18)37814-1
发表时间：
1988
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
E. Gruskin;R. Lloyd
通讯作者：
R. Lloyd
核酸内切酶 V 的突变影响蛋白质-蛋白质关联和靶位点定位。
影响因子：
2.9
作者：
Nickell,C;Lloyd,RS
通讯作者：
Lloyd,RS