A substrate recognition role for the [4Fe-4S]2+ cluster of the DNA repair glycosylase MutY.
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.
Porello
S L
SL
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Cannon
M J
MJ
David
S S
SS
D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D045647:DNA Glycosylases; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D013329:Structure-Activity Relationship
D007506:Iron-Sulfur Proteins; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase