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

Silvia L. Porello, Scott D. Williams, Heiko Kuhn, Mark L. Michaels, Sheila S. David

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)10684-10692
Number of pages9
JournalJournal of the American Chemical Society
Volume118
Issue number44
DOIs
StatePublished - Nov 6 1996

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ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Porello, S. L., Williams, S. D., Kuhn, H., Michaels, M. L., & David, S. S. (1996). Specific recognition of substrate analogs by the DNA mismatch repair enzyme MutY. Journal of the American Chemical Society, 118(44), 10684-10692. https://doi.org/10.1021/ja9602206