Effects of hydrogen bonding within a damaged base pair on the activity of wild type and DNA-intercalating mutants of human alkyladenine DNA glycosylase.
Human alkyladenine DNA glycosylase "flips" damaged DNA bases into its active site where excision occurs. Tyrosine 162 is inserted into the DNA helix in place of the damaged base and may assist in nucleotide flipping by "pushing" it. Mutating this DNA-intercalating Tyr to Ser reduces the DNA binding and base excision activities of alkyladenine DNA glycosylase to undetectable levels demonstrating that Tyr-162 is critical for both activities. Mutation of Tyr-162 to Phe reduces the single turnover excision rate of hypoxanthine by a factor of 4 when paired with thymine. Interestingly, when the base pairing partner for hypoxanthine is changed to difluorotoluene, which cannot hydrogen bond to hypoxanthine, single turnover excision rates increase by a factor of 2 for the wild type enzyme and about 3 to 4 for the Phe mutant. In assays with DNA substrates containing 1,N(6)-ethenoadenine, which does not form hydrogen bonds with either thymine or difluorotoluene, base excision rates for both the wild type and Phe mutant were unaffected. These results are consistent with a role for Tyr-162 in pushing the damaged base to assist in nucleotide flipping and indicate that a nucleotide flipping step may be rate-limiting for excision of hypoxanthine.
Vallur
Aarthy C
AC
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245, USA.
Feller
Joyce A
JA
Abner
Clint W
CW
Tran
Robert K
RK
Bloom
Linda B
LB
D020137:Base Pair Mismatch; D020029:Base Pairing; D004249:DNA Damage; D045647:DNA Glycosylases; D006801:Humans; D006860:Hydrogen Bonding; D007364:Intercalating Agents; D007700:Kinetics; D016296:Mutagenesis; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism
D007364:Intercalating Agents; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases