Structural Characterization of Heavy Metal Toxicity in a Human DNA Repair Glycosylase

Abstract

The DNA base excision repair pathway consists of several enzymes, some of which require metal cations for catalysis. Human Uracil DNA Glycosylase (hUNG), a DNA glycosylase responsible for the initial detection and removal of carcinogenic lesions from DNA, is a non-metal requiring enzyme. Using biochemical assays we showed that cadmium II (Cd), a known human carcinogen, inhibits hUNG activity in the micromolar range. To provide mechanistic insights for metal enzyme interactions, we employed computational approaches based on MD and QM calculations. Initially, our 1 μs explicit solvent Molecular Dynamics simulations using the AMBER force field showed Cd entering the hUNG pocket, interacting with the catalytic D64 residue. Subsequent simulation at the PM3 level showed that Cd additionally interacts with H67 and the peptide oxygen of P65 in the presence of a chloride ion. This position effectively replaces the catalytic water essential for hUNG activity. Further quantum mechanical refinement using M06L/QZVP ONIOM of Gaussian 09 on structures obtained from the PM3 simulations produced two distinct geometries of Cd in coordination 5. Both hUNG-Cd complexes are in agreement with metal sites found in a recent database of protein-metal complexes (MetalPDB). This work for the first time demonstrates a possible binding interaction between hUNG and toxic metal. In concert with our biochemical data we propose that metal inhibition of hUNG is due to replacement of the catalytic water, where a stable metal complex forms and prevents catalysis. The inhibition of hUNG by Cd will enhance the mutagenic and carcinogenic potentials of a variety of parental genotoxic compounds.