A Substrate Recognition Role for the [4Fe-4S]²⁺ Cluster of the DNA Repair Glycosylase MutY

Abstract

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.

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

“…However, the David laboratory has since performed several experiments to investigate the role of the cluster in this protein [36][37][38]. They have developed a method to remove reversibly the cluster from the protein and discovered that the cofactor is not necessary for protein folding nor does it contribute to the thermal stability of the protein.…”

DNA charge transport as a first step in coordinating the detection of lesions by repair proteins

“…Several DNA repair proteins contain redox-active [4Fe-4S] clusters that are not required for folding (12,13). Examples of these proteins from the BER pathway, EndoIII, and MutY, are activated toward oxidation as they bind DNA (14)(15)(16). While EndoIII effectively removes oxidized pyrimidines, the repair protein is found in very low copy number in E. coli (approximately 500 copies per cell) (17,18).…”

DNA-mediated charge transport for DNA repair

“…The function, if any, for these clusters remains undetermined, although the FCL has been proposed as a structural element, aiding in DNA binding (9,30,38). Interestingly, however, MutY is capable of folding without the cluster; the [4Fe4S] 2ϩ cluster adds no stability to the enzyme, but it is critical for substrate binding and catalysis (39). The solvent-accessible [4Fe4S] 2ϩ cluster of Endo III undergoes decomposition with ferricyanide and is resistant to reduction, with an estimated midpoint potential of ϽϪ600 mV for the [4Fe4S] 2ϩ/1ϩ couple (2,38).…”