Probing the Requirements for Recognition and Catalysis in Fpg and MutY with Nonpolar Adenine Isosteres

Anthony W. Francis, Sandra A. Helquist, Eric T. Kool, Sheila S. David

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

The Escherichia coli DNA repair enzymes Fpg and MutY are involved in the prevention of mutations resulting from 7,8-dihydro-8-oxo-2′ -deoxyguanosine (OG) in DNA. The nonpolar isosteres of 2′-deoxyadenosine, 4-methylbenzimidazole β-deoxynucleoside (B), and 9-methyl-1H-imidazo[4,5-b]pyridine β-deoxynucleoside (Q), were used to examine the importance of hydrogen bonding within the context of DNA repair. Specifically, the rate of base removal under single-turnover conditions by the MutY and Fpg glycosylases from duplexes containing OG:B and OG:Q mismatches, relative to OG:A mismatches, was evalulated. The reaction of Fpg revealed a 5- and 10-fold increase in rate of removal of OG from duplexes containing OG:B and OG:Q base pairs, respectively, relative to an OG:A mispair. These results suggest that the lack of the ability to hydrogen bond to the opposite base facilitates removal of OG. In contrast, adenine removal catalyzed by MutY was much more efficient from an OG:A mispair-containing duplex (k2 = 12 ± 2 min-1) compared to the removal of B from an OG:B duplex (kobs < 0.002 min-1). Surprisingly, MutY was able to catalyze base removal from the OG:Q-containing substrate (k2 = 1.2 ± 0.2 min-1). Importantly, the B and Q analogues are not deleterious to high-affinity DNA binding by MutY. In addition, the B and Q analogues are more susceptible to acid-catalyzed depurination illustrating that the enzyme-catalyzed mechanism is distinct from the nonenzymatic mechanism. Taken together, these results point to the importance of both N7 and N3 in the mechanism of adenine excision catalyzed by MutY.

Original languageEnglish (US)
Pages (from-to)16235-16242
Number of pages8
JournalJournal of the American Chemical Society
Volume125
Issue number52
DOIs
StatePublished - Dec 31 2003
Externally publishedYes

Fingerprint

Adenine
Catalysis
DNA Repair Enzymes
DNA
Hydrogen Bonding
Base Pairing
DNA Repair
Hydrogen
Escherichia coli
Mutation
Acids
Hydrogen bonds
Enzymes
Pyridine
Repair
Substrates
8-oxo-7-hydrodeoxyguanosine
mutY adenine glycosylase
1H-imidazo(4,5-b)pyridine
2'-deoxyadenosine

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Probing the Requirements for Recognition and Catalysis in Fpg and MutY with Nonpolar Adenine Isosteres. / Francis, Anthony W.; Helquist, Sandra A.; Kool, Eric T.; David, Sheila S.

In: Journal of the American Chemical Society, Vol. 125, No. 52, 31.12.2003, p. 16235-16242.

Research output: Contribution to journalArticle

Francis, Anthony W. ; Helquist, Sandra A. ; Kool, Eric T. ; David, Sheila S. / Probing the Requirements for Recognition and Catalysis in Fpg and MutY with Nonpolar Adenine Isosteres. In: Journal of the American Chemical Society. 2003 ; Vol. 125, No. 52. pp. 16235-16242.
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abstract = "The Escherichia coli DNA repair enzymes Fpg and MutY are involved in the prevention of mutations resulting from 7,8-dihydro-8-oxo-2′ -deoxyguanosine (OG) in DNA. The nonpolar isosteres of 2′-deoxyadenosine, 4-methylbenzimidazole β-deoxynucleoside (B), and 9-methyl-1H-imidazo[4,5-b]pyridine β-deoxynucleoside (Q), were used to examine the importance of hydrogen bonding within the context of DNA repair. Specifically, the rate of base removal under single-turnover conditions by the MutY and Fpg glycosylases from duplexes containing OG:B and OG:Q mismatches, relative to OG:A mismatches, was evalulated. The reaction of Fpg revealed a 5- and 10-fold increase in rate of removal of OG from duplexes containing OG:B and OG:Q base pairs, respectively, relative to an OG:A mispair. These results suggest that the lack of the ability to hydrogen bond to the opposite base facilitates removal of OG. In contrast, adenine removal catalyzed by MutY was much more efficient from an OG:A mispair-containing duplex (k2 = 12 ± 2 min-1) compared to the removal of B from an OG:B duplex (kobs < 0.002 min-1). Surprisingly, MutY was able to catalyze base removal from the OG:Q-containing substrate (k2 = 1.2 ± 0.2 min-1). Importantly, the B and Q analogues are not deleterious to high-affinity DNA binding by MutY. In addition, the B and Q analogues are more susceptible to acid-catalyzed depurination illustrating that the enzyme-catalyzed mechanism is distinct from the nonenzymatic mechanism. Taken together, these results point to the importance of both N7 and N3 in the mechanism of adenine excision catalyzed by MutY.",
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