4UQM

Crystal structure determination of uracil-DNA N-glycosylase (UNG) from Deinococcus radiodurans in complex with DNA - new insights into the role of the Leucine-loop for damage recognition and repair

Summary for 4UQM

• Entry DOI: 10.2210/pdb4uqm/pdb
• Descriptor: URACIL-DNA GLYCOSYLASE, 5'-D(*CP*CP*TP*AP*TP*CP*CP*AP*AAB*GP*TP*CP*TP*CP*CP*G)-3', 5'-D(*GP*CP*GP*GP*AP*GP*AP*CP*AP*TP*GP*GP*AP*CP*AP*G)-3', ... (6 entities in total)
• Functional Keywords: hydrolase-dna complex, base excision repair, radiation resistance, dna damage, dna repair, protein-dna complex, hydrolase/dna
• Biological source: DEINOCOCCUS RADIODURANS; More
• Cellular location: Cytoplasm : Q9RWH9
• Total number of polymer chains: 3
• Total formula weight: 37596.33

Authors

Pedersen, H.L.,Johnson, K.A.,McVey, C.E.,Leiros, I.,Moe, E. (deposition date: 2014-06-24, release date: 2015-08-12, Last modification date: 2024-01-10)

Primary citation

Pedersen, H.L.,Johnson, K.A.,McVey, C.E.,Leiros, I.,Moe, E.
Structure determination of uracil-DNA N-glycosylase from Deinococcus radiodurans in complex with DNA.
Acta Crystallogr. D Biol. Crystallogr., 71:2137-2149, 2015

PubMed 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.

PubMed: 26457437
DOI: 10.1107/S1399004715014157

Experimental method

X-RAY DIFFRACTION (1.35 Å)