SPECIFIC RECOGNITION OF SUBSTRATE-ANALOGS BY THE DNA MISMATCH REPAIR ENZYME MUTY

The DNA repair enzyme MutY plays an important role in the prevention o f DNA mutations caused by the oxidatively damaged lesion 7,8-dihydro-8 -oxo-2'-deoxyguanosine (OG) by removal of misincorporated adenine resi dues in OG:A mismatched base pairs using N-glycosylase activity. MutY also has glycosylase activity toward adenine in the mismatched base-pa irs G:A and C:A. We have investigated the interaction of MutY with DNA duplexes containing the 2'-deoxyadenosine (A) analogs 2'-deoxytuberci din (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 structur al properties. Thus, these derivatives will provide a method for formi ng a stable MutY-substrate analog complex amenable to structural and b iochemical investigation. We find that oligonucleotide duplexes contai ning OG/G:F and OG/G:Z base-pairs are not substrates for MutY as expec ted. Using a gel retardation method to measure relevant K-d values, we determined that MutY has an increased association with duplexes conta ining OG/G:F and OG/G:Z base-pairs over their OG/G:C counterparts. Int erestingly, MutY has a higher affinity for the F-containing duplexes t han the Z counterparts. Additionally, MutY binds to the OG:F and G:F d uplexes with a similar, albeit lower, affinity as the substrate OG:A a nd G:A duplexes. In footprinting experiments using methidiumpropyl-EDT A-Fe(II), a region of the duplex surrounding the OG:F base-pair is obs erved which is protected by MutY from hydroxyl radical cleavage. These results provide additional evidence for specific recognition of the O G:F base-pair within the DNA duplex. Furthermore, these results also i llustrate the utility of OG:F duplexes for providing information regar ding the MutY-mismatched DNA complex which could not be obtained with the normal OG:A substrate since a footprint on both strands of the dup lex could only be observed with the OG:F-containing duplex. These subs trate analog duplexes will provide avenues for structural analysis of the MutY-mismatched DNA complex and for investigating the properties o f the unusual [4Fe-4S] center in MutY.