title: Structure determination of uracil-DNA N-glycosylase from Deinococcus radiodurans in complex with DNA.
abstract: Uracil-DNA N-glycosylase (UNG) is a DNA-repair enzyme in the base-excision repair (BER) pathway which removes uracil from DNA. Here, the crystal structure of UNG from the extremophilic bacterium Deinococcus radiodurans (DrUNG) in complex with DNA is reported at a resolution of 1.35 Å. Prior to the crystallization experiments, the affinity between DrUNG and different DNA oligonucleotides was tested by electrophoretic mobility shift assays (EMSAs). As a result of this analysis, two 16 nt double-stranded DNAs were chosen for the co-crystallization experiments, one of which (16 nt AU) resulted in well diffracting crystals. The DNA in the co-crystal structure contained an abasic site (substrate product) flipped into the active site of the enzyme, with no uracil in the active-site pocket. Despite the high resolution, it was not possible to fit all of the terminal nucleotides of the DNA complex into electron density owing to disorder caused by a lack of stabilizing interactions. However, the DNA which was in contact with the enzyme, close to the active site, was well ordered and allowed detailed analysis of the enzyme-DNA interaction. The complex revealed that the interaction between DrUNG and DNA is similar to that in the previously determined crystal structure of human UNG (hUNG) in complex with DNA [Slupphaug et al. (1996). Nature (London), 384, 87-92]. Substitutions in a (here defined) variable part of the leucine loop result in a shorter loop (eight residues instead of nine) in DrUNG compared with hUNG; regardless of this, it seems to fulfil its role and generate a stabilizing force with the minor groove upon flipping out of the damaged base into the active site. The structure also provides a rationale for the previously observed high catalytic efficiency of DrUNG caused by high substrate affinity by demonstrating an increased number of long-range electrostatic interactions between the enzyme and the DNA. Interestingly, specific interactions between residues in the N-terminus of a symmetry-related molecule and the complementary DNA strand facing away from the active site were also observed which seem to stabilize the enzyme-DNA complex. However, the significance of this observation remains to be investigated. The results provide new insights into the current knowledge about DNA damage recognition and repair by uracil-DNA glycosylases.
authors.affiliation: The Norwegian Structural Biology Center (NorStruct), Department of Chemistry, UiT - The Arctic University of Norway, 9037 Tromsø, Norway.
authors.forename: Hege Lynum
authors.initials: HL
authors.lastname: Pedersen
authors.affiliation: The Norwegian Structural Biology Center (NorStruct), Department of Chemistry, UiT - The Arctic University of Norway, 9037 Tromsø, Norway.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Johnson
authors.affiliation: Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal.
authors.forename: Colin E
authors.initials: CE
authors.lastname: McVey
authors.affiliation: The Norwegian Structural Biology Center (NorStruct), Department of Chemistry, UiT - The Arctic University of Norway, 9037 Tromsø, Norway.
authors.forename: Ingar
authors.initials: I
authors.lastname: Leiros
authors.affiliation: The Norwegian Structural Biology Center (NorStruct), Department of Chemistry, UiT - The Arctic University of Norway, 9037 Tromsø, Norway.
authors.forename: Elin
authors.initials: E
authors.lastname: Moe
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D034301:Deinococcus / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D051981:Uracil-DNA Glycosidase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004247:DNA; D051981:Uracil-DNA Glycosidase
keywords: Deinococcus radiodurans; base-excision repair; protein–DNA co-crystal structure; uracil-DNA N-glycosylase