Mutants of the Base Excision Repair Glycosylase, Endonuclease III: DNA Charge Transport as a First Step in Lesion Detection
Endonuclease III (EndoIII) is a base excision repair glycosylase that targets damaged pyrimidines and contains a [4Fe-4S] cluster. We have proposed a model where BER proteins that contain redox-active [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in the detection of DNA lesions. Here, several mutants of EndoIII were prepared to probe their efficiency of DNA/protein charge transport. Cyclic voltammetry experiments on DNA-modified electrodes show that aromatic residues F30, Y55, Y75, and Y82 help mediate charge transport between DNA and the [4Fe-4S] cluster. On the basis of circular dichroism studies to measure protein stability, mutations at residues W178 and Y185 are found to destabilize the protein; these residues may function to protect the [4Fe-4S] cluster. Atomic force microscopy studies furthermore reveal a correlation in the ability of mutants to carry out protein/DNA CT and their ability to relocalize onto DNA strands containing a single base mismatch; EndoIII mutants that are defective in carrying out DNA/protein CT do not redistribute onto mismatch-containing strands, consistent with our model. These results demonstrate a link between the ability of the repair protein to carry out DNA CT and its ability to relocalize near lesions, thus pointing to DNA CT as a key first step in the detection of base damage in the genome.
© 2011 American Chemical Society. Received: March 2, 2011; revised: May 9, 2011; published: June 9, 2011. We are grateful to the NIH (GM49216) for their support and to NDSEG and the Rosen fund for fellowship support to C.A.R. The authors gratefully acknowledge H. B. Gray, D. K. Newman, A. K. Boal, and J. C. Genereux for helpful discussions. T. J. Ge and A. Parisian provided technical assistance, and E. D. Olmon helped prepare figures. We are also grateful to the Beckman Institute MMRC for AFM instrumentation.
Accepted Version - nihms302613.pdf
Supplemental Material - bi2003179_si_001.pdf