Porello S L, Cannon M J, David S S
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Biochemistry. 1998 May 5;37(18):6465-75. doi: 10.1021/bi972433t.
The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7, 8-dihydro-8-oxo-2'-deoxyguanosine: 2'-deoxyadenosine (OG:A) mismatches in DNA [Michaels et al. (1992) Proc. Natl. Acad. Sci. U.S. A. 89, 7022-7025]. MutY prevents mutations due to misincorporation of A opposite OG during DNA replication by removing the adenine base. This enzyme has significant sequence homology with the [4Fe-4S]2+ cluster-containing DNA repair enzyme, endonuclease III [Michaels et al. (1990) Nucleic AcidsRes. 18, 3841-3845]. In the present study, we have investigated the importance of cluster assembly in folding of MutY. MutY was denatured and then refolded in the presence or absence of ferrous and sulfide ions. Denatured MutY can refold in the presence of ferrous and sulfide ions to provide active enzyme. This suggests the cluster can self-assemble and that this process is facile in vitro. Interestingly, CD spectra and Tm measurements of MutY refolded with and without ferrous and sulfide ions are essentially identical, implying that assembly of the cluster is not required for MutY folding. Additionally, Tm measurements indicated that the [4Fe-4S]2+ cluster does not contribute significantly to the overall thermal stability of MutY. Refolded forms of MutY which lack the cluster are unable to perform the adenine glycosylase function and bind to DNA. However, these inactive folded forms regain activity by addition of ferrous and sulfide ions. This indicates that the Fe-S cluster may have a superficial location, allowing for its assembly after folding. More importantly, these results provide evidence that the presence of the [4Fe-4S]2+ cluster is critical for the specific recognition of substrate DNA necessary for the adenine glycosylase activity of MutY.
大肠杆菌DNA修复酶MutY在识别和修复DNA中的7,8 - 二氢 - 8 - 氧代 - 2'-脱氧鸟苷：2'-脱氧腺苷（OG:A）错配中起重要作用[迈克尔等人（1992年）《美国国家科学院院刊》89, 7022 - 7025]。MutY通过去除腺嘌呤碱基来防止DNA复制过程中由于OG对面误掺入A而导致的突变。这种酶与含[4Fe - 4S]2+簇的DNA修复酶核酸内切酶III具有显著的序列同源性[迈克尔等人（1990年）《核酸研究》18, 3841 - 3845]。在本研究中，我们研究了簇组装在MutY折叠中的重要性。MutY被变性，然后在有或没有亚铁离子和硫化物离子的情况下重新折叠。变性的MutY在亚铁离子和硫化物离子存在下可以重新折叠以提供活性酶。这表明簇可以自组装，并且该过程在体外很容易进行。有趣的是，在有和没有亚铁离子和硫化物离子的情况下重新折叠的MutY的圆二色光谱和熔解温度测量结果基本相同，这意味着簇的组装对于MutY折叠不是必需的。此外，熔解温度测量表明[4Fe - 4S]2+簇对MutY的整体热稳定性没有显著贡献。缺乏簇的MutY重新折叠形式无法执行腺嘌呤糖基化酶功能并与DNA结合。然而，这些无活性的折叠形式通过添加亚铁离子和硫化物离子恢复活性。这表明Fe - S簇可能位于表面位置，可以在折叠后组装。更重要的是，这些结果提供了证据，表明[4Fe - 4S]2+簇的存在对于MutY腺嘌呤糖基化酶活性所需的底物DNA的特异性识别至关重要。
