Cold adaptation and molecular design of cod Uracil DNA glycosylase

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Project leader: Professor Arne o. Smalås and Professor Nils Peder Willassen

Uracil DNA glycosylase (UDG) is a DNA repair enzyme in the base excision repair pathway which removes uracil from the DNA strand. The enzyme, which is the first in the base excision repair pathway, catalyzes the hydrolysis of promutagenic uracil residues from single- or double-stranded DNA. The crystal structure of the catalytic domain of UDG from several species are known (e.g. human, herpes simplex virus and E.coli)

The project uses cod Uracil-DNA glycosylase (cUNG) as a model system to study of DNA recognition and cold activity. Cod UNG possesses typical cold adaptation features such as increased catalytic efficiency and reduced temperature stability compared to human UNG. The crystal structure of cod UNG shows similarity to the structures of human, herpes simplex virus and E.coli UNG. Furthermore, in the order of 30 mutants has so far been constructed and characterized on various levels. Characterization of mutants of cUNG in combination with structure analysis indicates a highly fine-tuned mechanism responsible for the increased catalytic efficiency. Analysis of mutants with substitutions in loop regions and alterations of the surface charge indicate that an increased local flexibility and an optimisation of the electrostatic potential cause the increased activity of the enzyme. These observations are further explored by the use of crystal structure analysis combined with theoretical calculations on cUNG-DNA/inhibitor complexes. Crystal structure analysis and theoretical calculations are applied on both complexes with native enzyme and on a series of mutants. The results from the study are expected to provide increased insight into the molecular basis of protein-DNA interaction and cold activity.