ISOLATION AND CHARACTERIZATION OF ENDONUCLEASE-VIII FROM ESCHERICHIA-COLI
ISOLATION AND CHARACTERIZATION OF ENDONUCLEASE-VIII FROM ESCHERICHIA-COLI
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DOI：
10.1021/bi00171a028
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发表时间：
1994-02-08
期刊：
影响因子：
2.9
通讯作者：
WALLACE, SS
中科院分区：
文献类型：
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作者：
MELAMEDE, RJ;HATAHET, Z;WALLACE, SS
DNA内切酶VIII是从大肠杆菌中通过FPLC(1)纯化得到的一种新的DNA修复酶。该酶存在于含有或不含核酸内切酶III的菌株中，经S-琼脂糖、Mono S、苯基-超糖和Superose 12 FPLC的径向流动分离纯化。对内切酶性质的研究表明，它与内切酶III有许多相似之处。含有胸腺嘧啶二醇、二氢胸腺嘧啶、β-尿苷异丁酸、尿素残基或AP位点的DNA被该酶切割，而含有还原AP位点的DNA则不被切割。对含有损伤DNA的酶消化产物进行高效液相分析表明，内切酶VIII以游离碱的形式释放胸腺嘧啶、乙二醇和二氢胸腺嘧啶。综上所述，这些数据表明，内切酶VIII同时含有N-糖基酶和AP裂解酶活性。与这一观点一致的是，含有AP位点或胸腺嘧啶二醇的DNA被内切酶VIII酶切后不是大肠杆菌DNA聚合酶I的良好底物，这表明内切酶VIII在病变的3‘端划伤了含有损伤的DNA。此外，由于含有胸腺嘧啶二醇的DNA在用内切酶VIII处理后没有释放单磷酸，因此该酶似乎不具有核酸外切酶活性。在75 mM的氯化钠或5 mM的氯化镁中酶活最高。用Superose FPLC和Sephadex对内切酶进行分析，得到天然相对分子质量分别为28000和30000 Da。结合活性凝胶分析，其相对分子质量约为29000 Da。此外，从SDS-PAGE中推测的活性条带的复性产生了一种活性酶。
Endonuclease VIII, a novel presumptive DNA repair enzyme, was isolated from Escherichia coli by FPLC(1) purification. The enzyme was found in strains that contained or lacked endonuclease III and was purified by radial flow S-Sepharose, Mono S, phenyl-Superose, and Superose 12 FPLC. Examination of the properties of endonuclease VIII showed it to have many similarities to endonuclease III. DNA containing thymine glycol, dihydrothymine, beta-ureidoisobutyric acid, urea residues, or AP sites was incised by the enzyme; however, DNA containing reduced AP sites was not. HPLC analysis of the products formed by exhaustive enzymatic digestion of damage-containing DNA showed that endonuclease VIII released thymine glycol and dihydrothymine as free bases. Taken together, these data suggest that endonuclease VIII contains both N-glycosylase and AP lyase activities. Consistent with this idea, DNA containing AP sites or thymine glycols, that was enzymatically nicked by endonuclease VIII was not a good substrate for E. coli DNA polymerase I, suggesting that endonuclease VIII nicks damage-containing DNA on the 3' side of the lesion. Also, since monophosphates were not released after treating thymine glycol-containing DNA with endonuclease VIII, the enzyme does not appear to have exonuclease activity. The enzyme activity was maximal in 75 mM NaCl or 5 mM MgCl2. Analysis of endonuclease VIII by both Superose FPLC and Sephadex yielded native molecular masses of 28 000 and 30 000 Da, respectively. SDS-PAGE, in conjunction with activity gel analysis, gave a molecular mass of about 29 000 Da. Furthermore, renaturation of the putative active band from SDS-PAGE gave rise to an active enzyme.