Specificity and catalytic mechanism in family 5 uracil DNA glycosylase.
UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O2 of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb relies on multiple catalytic residues to facilitate its excision of hypoxanthine and xanthine. This study underscores the structural and functional diversity in the UDG superfamily.
From the Department of Genetics and Biochemistry, South Carolina Experiment Station and.
Bo
B
Xia
the Department of Chemistry, Clemson University, Clemson, South Carolina 29634.
Yinling
Y
Liu
From the Department of Genetics and Biochemistry, South Carolina Experiment Station and.
Wei
W
Li
the Department of Chemistry, Clemson University, Clemson, South Carolina 29634.
Allyn R
AR
Brice
the Department of Chemistry, Clemson University, Clemson, South Carolina 29634.
Brian N
BN
Dominy
From the Department of Genetics and Biochemistry, South Carolina Experiment Station and wgc@clemson.edu.
Weiguo
W
Cao
D000595:Amino Acid Sequence; D002384:Catalysis; D004260:DNA Repair; D019271:Hypoxanthine / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019820:Xanthine / Q000378:metabolism
D019820:Xanthine; D019271:Hypoxanthine; D014498:Uracil; D051981:Uracil-DNA Glycosidase
DNA Damage; DNA Glycosylase; DNA Repair; Deamination; Hypoxanthine; Molecular Modeling; Mutagenesis; Nucleic Acid Enzymology; Uracil; Xanthine