Substrate recognition by Escherichia coli MutY using substrate analogs.

The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2"-deoxyguanosine (OG):A and G:A mispairs in DNA. Our approach toward understanding recognition and processing of DNA damage by MutY has been to use substrate analogs that retain the recognition properties of the substrate mispair but are resistant to the glycosylase activity of MutY. This approach provides stable MutY-DNA complexes that are amenable to structural and biochemical characterization. In this work, the interaction of MutY with the 2"-deoxyadenosine analogs 2"-deoxy-2"-fluoroadenosine (FA), 2"-deoxyaristeromycin (R) and 2"-deoxyformycin A (F) was investigated. MutY binds to duplexes containing the FA, R or F analogs opposite G and OG within DNA with high affinity; however, no enzymatic processing of these duplexes is observed. The specific nature of the interaction of MutY with an OG:FA duplex was demonstrated by MPE-Fe(II) hydroxyl radical footprinting experiments which showed a nine base pair region of protection by MutY surrounding the mispair. DMS footprinting experiments with an OG:A duplex revealed that a specific G residue located on the OG-containing strand was protected from DMS in the presence of MutY. In contrast, a G residue flanking the substrate analogs R, F or FA was observed to be hypersensitive to DMS in the presence of MutY. These results suggest a major conformational change in the DNA helix upon binding of MutY that exposes the substrate analog-containing strand. This finding is consistent with a nucleotide flipping mechanism for damage recognition by MutY. This work demonstrates that duplex substrates for MutY containing FA, R or F instead of A are excellent substrate mimics that may be used to provide insight into the recognition by MutY of damaged and mismatched base pairs within DNA.

Single-turnover and pre-steady-state kinetics of the reaction of the adenine glycosylase MutY with mismatch-containing DNA substrates.

The DNA repair enzyme MutY plays an important role in the prevention of DNA mutations resulting from the presence of the oxidatively damaged lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) in DNA by the removal of misincorporated adenine residues in OG:A mispairs. MutY also exhibits adenine glycosylase activity toward adenine in G:A and C:A mismatches, although the importance of this activity in vivo has not been established. We have investigated the kinetic properties of MutY's glycosylase activity with OG:A and G:A containing DNA duplexes. Our results indicate that MutY's processing of these two substrates is distinctly different. By using single-turnover experiments, the intrinsic rate for adenine removal by MutY from an OG:A substrate was found to be at least 6-fold faster than that from the corresponding G:A substrate. However, under conditions where [MutY] << [DNA], OG:A substrates are not quantitatively converted to product due to the inefficient turnover resulting from slow product release. In contrast, with a G:A substrate MutY's dissociation from the corresponding product is more facile, such that complete conversion of the substrate to product can be achieved under similar conditions. The kinetic results illustrate that the glycosylase reaction catalyzed by MutY has significant differences depending on the characteristics of the substrate. The lingering of MutY with the product of its reaction with OG:A mispairs may be biologically significant to prevent premature removal of OG. Thus, this approach is providing insight into factors that may be influencing the repair of damaged and mismatched DNA in vivo by base-excision repair glycosylases.

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.