Probing the requirements for recognition and catalysis in Fpg and MutY with nonpolar adenine isosteres.
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 beta-deoxynucleoside (B), and 9-methyl-1H-imidazo[4,5-b]pyridine beta-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 (k(obs) < 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.
Francis
Anthony W
AW
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah, 84112, USA.
Helquist
Sandra A
SA
Kool
Eric T
ET
David
Sheila S
SS
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D020137:Base Pair Mismatch; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D011684:Purine Nucleosides / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D007093:Imidazoles; D011684:Purine Nucleosides; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C437225:adenine glycosylase; C061738:mutY adenine glycosylase; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D003849:Deoxyguanosine; D000225:Adenine