title: A structural determinant in the uracil DNA glycosylase superfamily for the removal of uracil from adenine/uracil base pairs.
abstract: The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-specific uracil DNA glycosylase (MUG) from Escherichia coli is known to exhibit glycosylase activity on three mismatched base pairs, T/U, G/U and C/U. Family 1 uracil N-glycosylase (UNG) from E. coli is an extremely efficient enzyme that can remove uracil from any uracil-containing base pairs including the A/U base pair. Here, we report the identification of an important structural determinant that underlies the functional difference between MUG and UNG. Substitution of a Lys residue at position 68 with Asn in MUG not only accelerates the removal of uracil from mismatched base pairs but also enables the enzyme to gain catalytic activity on A/U base pairs. Binding and kinetic analysis demonstrate that the MUG-K68N substitution results in enhanced ground state binding and transition state interactions. Molecular modeling reveals that MUG-K68N, UNG-N123 and family 5 Thermus thermophiles UDGb-A111N can form bidentate hydrogen bonds with the N3 and O4 moieties of the uracil base. Genetic analysis indicates the gain of function for A/U base pairs allows the MUG-K68N mutant to remove uracil incorporated into the genome during DNA replication. The implications of this study in the origin of life are discussed.
authors.affiliation: Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, 049 Life Sciences Facility, 190 Collings Street, Clemson, SC 29634, USA.
authors.forename: Dong-Hoon
authors.initials: DH
authors.lastname: Lee
authors.affiliation: 367 Hunter Laboratories, Department of Chemistry, Clemson University, Clemson, SC 29634, USA.
authors.forename: Yinling
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, 049 Life Sciences Facility, 190 Collings Street, Clemson, SC 29634, USA.
authors.forename: Hyun-Wook
authors.initials: HW
authors.lastname: Lee
authors.affiliation: Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, 049 Life Sciences Facility, 190 Collings Street, Clemson, SC 29634, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Xia
authors.affiliation: 367 Hunter Laboratories, Department of Chemistry, Clemson University, Clemson, SC 29634, USA.
authors.forename: Allyn R
authors.initials: AR
authors.lastname: Brice
authors.affiliation: Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, 049 Life Sciences Facility, 190 Collings Street, Clemson, SC 29634, USA.
authors.forename: Sung-Hyun
authors.initials: SH
authors.lastname: Park
authors.affiliation: Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, 049 Life Sciences Facility, 190 Collings Street, Clemson, SC 29634, USA.
authors.forename: Hunter
authors.initials: H
authors.lastname: Balduf
authors.affiliation: 367 Hunter Laboratories, Department of Chemistry, Clemson University, Clemson, SC 29634, USA.
authors.forename: Brian N
authors.initials: BN
authors.lastname: Dominy
authors.affiliation: Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, 049 Life Sciences Facility, 190 Collings Street, Clemson, SC 29634, USA wgc@clemson.edu.
authors.forename: Weiguo
authors.initials: W
authors.lastname: Cao
mesh_terms: D000225:Adenine / Q000378:metabolism; D019943:Amino Acid Substitution; D020137:Base Pair Mismatch; D020029:Base Pairing; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D014498:Uracil / Q000737:chemistry* / Q000378:metabolism; D051981:Uracil-DNA Glycosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D014498:Uracil; D051981:Uracil-DNA Glycosidase; D000225:Adenine