Specific recognition of substrate analogs by the DNA mismatch repair enzyme MutY

The DNA repair enzyme MutY plays an important role in the prevention of DNA mutations caused by the oxidatively damaged lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removal of misincorporated adenine residues in OG:A mismatched base pairs using N-glycosylase activity. MutY also has glycosylase activity toward adenine in the mismatched base-pairs G:A and C:A. We have investigated the interaction of MutY with DNA duplexes containing the 2'-deoxyadenosine (A) analogs 2'-deoxytubercidin (7-deaza-2'-deoxyadenosine, Z) and 2'deoxyformycin A (F). Both F and Z should effectively mimic the recognition properties of A but be resistant to the glycosylase activity of MutY, owing to their structural properties. Thus, these derivatives will provide a method for forming a stable MutY-substrate analog complex amenable to structural and biochemical investigation. We find that oligonucleotide duplexes containing OG/G:F and OG/G:Z base-pairs are not substrates for MutY as expected. Using a gel retardation method to measure relevant K(d) values, we determined that MutY has an increased association with duplexes containing OG/G:F and OG/G:Z base-pairs over their OG/G:C counterparts. Interestingly, MutY has a higher affinity for the F-containing duplexes than the Z counterparts. Additionally, MutY binds to the OG:F and G:F duplexes with a similar, albeit lower, affinity as the substrate OG:A and G:A duplexes. In footprinting experiments using methidiumpropyl-EDTA-Fe(II), a region of the duplex surrounding the OG:F base-pair is observed which is protected by MutY from hydroxyl radical cleavage. These results provide additional evidence for specific recognition of the OG:F base-pair within the DNA duplex. Furthermore, these results also illustrate the utility of OG:F duplexes for providing information regarding the MutY-mismatched DNA complex which could not be obtained with the normal OG:A substrate since a footprint on both strands of the duplex could only be observed with the OG:F containing duplex. These substrate analog duplexes will provide avenues for structural analysis of the MutY-mismatched DNA complex and for investigating the properties of the unusual [4Fe-4S] center in MutY.