Abstract
Toxic metals are known to inhibit DNA repair but the underlying mechanisms of inhibition are still not fully understood. DNA repair enzymes such as human uracil-DNA glycosylase (hUNG) perform the initial step in the base excision repair (BER) pathway. In this work, we showed that cadmium [Cd(II)], a known human carcinogen, inhibited all activity of hUNG at 100 μM. Computational analyses based on 2 μs equilibrium, 1.6 μs steered molecular dynamics (SMD), and QM/MM MD determined that Cd(II) ions entered the enzyme active site and formed close contacts with both D145 and H148, effectively replacing the catalytic water normally found in this position. Geometry refinement by density functional theory (DFT) calculations showed that Cd(II) formed a tetrahedral structure with D145, P146, H148, and one water molecule. This work for the first time reports Cd(II) inhibition of hUNG which was due to replacement of the catalytic water by binding the active site D145 and H148 residues. Comparison of the proposed metal binding site to existing structural data showed that D145:H148 followed a general metal binding motif favored by Cd(II). The identified motif offered structural insights into metal inhibition of other DNA repair enzymes and glycosylases.
Highlights
IntroductionIn contrast to the above glycosylases, divalent metals play an essential role in the catalytic reactions of the other BER enzymes
Determining the ability of metal ions to inhibit glycosylases is critical for understanding mechanisms responsible for DNA repair deficiency
The work by us and others clearly demonstrated that toxic metals inhibit DNA repair, there is no structural data available for specific details on protein-metal interactions
Summary
In contrast to the above glycosylases, divalent metals play an essential role in the catalytic reactions of the other BER enzymes These enzymes include apurinic/apyrimidinic endonuclease (APE1) and the A/G-mismatch-specific adenine glycosylases MutY/Mig. It was proposed that the inhibitory effect of divalent metals on APE1 may be associated with their specific interaction with the active site residues[26]. Glycosidic cleavage produces 5-hydroxy ribose and an apurinic (AP) site in DNA Generation of such AP sites initiates the BER pathway, effectively making glycosylases the key to BER. The work by us and others clearly demonstrated that toxic metals inhibit DNA repair, there is no structural data available for specific details on protein-metal interactions.
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