Recognition and repair of the CC-1065-(N3-adenine)-DNA adduct by the UVRABC nucleases.
Recognition and repair of the CC-1065-(N3-adenine)-DNA adduct by the UVRABC nucleases.
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UVRABC 核酸酶对 CC-1065-(N3-腺嘌呤)-DNA 加合物的识别和修复。
DOI：
10.1021/bi00403a009
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Hurley,LH
中科院分区：
文献类型：
--
作者：
Tang,MS;Lee,CS;Doisy,R;Ross,L;Needham-VanDevanter,DR;Hurley,LH
修订稿于 1987 年 10 月 9 日收到 摘要：UVRABC 核酸酶对螺旋稳定且相对不扭曲的 CC-1065-(N3-腺嘌呤)-DNA 加合物的识别和修复已通过转染测定用 0X174 RFI DNA 在体内进行了研究，并在体外通过来自 M13mpl 的 117 个碱基对片段中的定点加合物进行了研究。 CC-1065 是由泽兰链霉菌 (Streptomyces zelensis) 产生的强效抗肿瘤抗生素，通过腺嘌呤的 N3 结合在 DNA 小沟内。与紫外线或 7V-乙酰氧基-2-（乙酰氨基）芴引起的 DNA 螺旋不稳定和扭曲修饰相反，CC-1065 提高了 DNA 的熔点并降低了 SI 核酸酶活性。使用病毒DNA-大肠杆菌转染系统，我们发现uvrA、uvrB和uvrC基因（编码UV和NAAAF诱导的DNA损伤的主要切除修复蛋白）也参与CC-1065-DNA加合物的修复。相比之下，已发现参与紫外线损伤修复的uvrD基因产物，对修复CC-1065-DNA加合物没有作用。纯化的 UVRA、UVRB 和 UVRC 蛋白必须协同工作才能切割药物修饰的 0X174 RFI DNA。使用来自M13mpl的定点和多个CC-1065修饰的(Mspl-BstNl) 117个碱基对片段，我们发现UVRABC核酸酶在药物修饰链上CC-1065-DNA加合物的5'侧的第八个磷酸二酯键处切割。酶不会切割非共价修饰的链。在低药物结合率下，在 (MspI-BstNI) 117 个碱基对片段 GATTA*、GGAAA*、GATAA* 和 TTTTA*（* 表示共价修饰的腺嘌呤）中鉴定的四个 CC-1065 结合位点中，只有高亲和力结合位点 GATTA* 处的加合物被 UVRABC 核酸酶切割。根据 DNase I 切割模式确定，CC-1065 对局部 DNA 结构的影响在这些位点之间没有明显差异。在高药物结合率下，确定了第五个药物结合位点 AGCTA*。在此浓度下，UVRABC 核酸酶无法切割这五种 CC-1065-DNA 加合物中的任何一种。多种加合物的DNA序列和/或螺旋稳定作用可以决定UVRABC核酸酶对药物-DNA加合物的识别和/或切割。这些结果与 CC-1065-DNA 加合物的结构以及药物结合对局部 DNA 结构的影响进行了讨论。大肠杆菌细胞中的 uvrA、uvrB 和 uvrC 基因控制由 UV1 辐射和大量化学致癌物和药物（如 NAAAF、苯并[a]芘二醇环氧化物、m-铂和补骨脂素）诱导的 DNA 损伤切除修复的初始步骤（Seeberg 等，1983；Sancar 等，1985；Beck 等， 1985）。这些试剂会引起 DNA 螺旋不同程度的变形；这些试剂损伤的 DNA 对 SI 核酸酶消化更敏感（Grunberger & Weinstein，1978），并且还表现出降低的解链温度（Trifonov 等，1968；Fuchs & Duane，1974；Pulkrabek 等，1978）。纯化的 uvrA、uvrB 和 uvrC 基因产物已被证明可以协同作用，切割受损碱基的 5' 侧（第八个磷酸二酯键）和 3' 侧（第五或第四个磷酸二酯键），例如环丁烷二嘧啶、(6-4) 光产物、顺铂加合物和 dG-C8-AAF。 CC-1065 是一种由泽兰链霉菌 (Streptomyces zelensis) 产生的有效抗肿瘤抗生素 [Hanka 等人，1978，有关最新评论，请参见 Hurley 和 Needham-VanDevanter (1986) 和 Reynolds 等人 (1986)]。 CC-1065 的三个苯并二吡咯亚基
Revised Manuscript Received October 9, 1987 abstract: The recognition and repair of the helix-stabilizing and relatively nondistortive CC-1065-(N3-adenine)-DNA adduct by UVRABC nuclease has been investigated bothin vivo with 0X174 RFI DNA by a transfection assay and in vitro by a site-directed adduct in a 117 base pair fragment from M13mpl. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis which binds within the minor groove of DNA through N3 of adenine. In contrast to the helix-destabilizing and distortive modifications of DNA caused by ultraviolet light or 7V-acetoxy-2-(acetylamino) fluorene, CC-1065 increases the melting point of DNA and decreases the SI nuclease activity. Using a viral DNA-Escherichia coli transfection system, we have found that the uvrA, uvrB, and uvrC genes, which code for the major excision repair proteins for UV-and NAAAF-induced DNA damage, are also involved in the repair of CC-1065-DNA adducts. In contrast, the uvrD gene product, which has been found to be involved in the repair of UV damage, has no effect in repairing CC-1065-DNA adducts. Purified UVRA, UVRB, and UVRC proteins must work in concert to incise the drug-modified 0X174 RFI DNA. Using a site-directed and multiple CC-1065 modified (Mspl-BstNl) 117 base pair fragment from M13mpl, we have foundthat UVRABC nuclease incises at the eighth phosphodiester bond on the 5'side of the CC-1065-DNA adduct on the drug-modified strand. The enzymes do not cut the noncovalently modified strand. At low drug binding ratios, of the four CC-1065 binding sites identified in the (MspI-BstNI) 117 base pair fragment, GATTA*, GGAAA*, GATAA*, and TTTTA*(* indicates the covalently modified adenine), only the adduct at the high-affinity binding site, GATTA*, is incised by the UVRABC nucleases. No difference in the effect of CC-1065 on local DNA structure, as determined by the DNase I cleavage pattern, was evident among thesesites. At high drug binding ratios, a fifth drug binding site, AGCTA*, is identified. At this concentration UVRABC nucleases are unable to incise any of these five CC-1065-DNA adducts. The DNA sequence and/or helix-stabilizing effect of multiple adducts may determine the recognition and/or incision of the drug-DNA adduct by UVRABC nuclease. These results are discussed in relation to the structure of the CC-1065-DNA adduct and the effect of drug binding on local DNA structure. e uvrA, uvrB, and uvrC genes in Escherichia coli cells control the initial steps of excision repair of DNA damage induced by UV1 radiation and by bulky chemical carcinogens and drugs such as NAAAF, benzo [a] pyrenediol epoxide, m-platinum, and psoralen (Seeberg et al., 1983; Sancar et al., 1985; Beck et al., 1985). These agents induce various extents of deformation in DNA helix; DNA damaged by these agents is more sensitive to SI nuclease digestion (Grunberger & Weinstein, 1978) and also exhibits a decreased melting temperature (Trifonov et al., 1968; Fuchs & Duane, 1974; Pulkrabek et al., 1978). Purified uvrA, uvrB, and uvrC gene products have been shown to work in concert in incising both the 5'side (eighth phosphodiester bond) and the 3'side (fifth or fourth phosphodiester bond) of the damaged base (s), such as cyclobutane dipyrimidines,(6-4) photoproducts, cw-platinum adducts, and dG-C8-AAF. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis [Hanka et al., 1978, for recent reviews, see Hurley and Needham-VanDevanter (1986) and Reynolds et al.(1986)]. The three benzodipyrrole subunits of CC-1065