A SUBSTRATE RECOGNITION ROLE FOR THE [4FE-4S](2-REPAIR GLYCOSYLASE MUTY() CLUSTER OF THE DNA)

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 t o 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 II I [Michaels et al. (1990) Nucleic Acids Res. 18, 3841-3845]. In the pr esent study, we have investigated the importance of cluster assembly i n folding of MutY. MutY was denatured and then refolded in the presenc e or absence of ferrous and sulfide ions. Denatured MutY can refold in the presence of ferrous and sulfide ions to provide active enzyme. Th is suggests the cluster can self-assemble and that this process is fac ile in vitro. Interestingly, CD spectra and T-m measurements of MutY r efolded with and without ferrous and sulfide ions are essentially iden tical, implying that assembly of the cluster is not required for MutY folding. Additionally, T-m measurements indicated that the [4Fe-4S](2) cluster does not contribute significantly to the overall thermal sta bility of MutY. Refolded forms of MutY which lack the cluster are unab le to perform the adenine glycosylase function and bind to DNA. Howeve r, these inactive folded forms regain activity by addition of ferrous and sulfide ions. This indicates that the Fe-S cluster may have a supe rficial location, allowing for its assembly after folding. More import antly, these results provide evidence that the presence of the [4Fe-4S ](2+) cluster is critical for the specific recognition of substrate DN A necessary for the adenine glycosylase activity of MutY.