title: Mutants of the base excision repair glycosylase, endonuclease III: DNA charge transport as a first step in lesion detection.
abstract: 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.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technolog, Pasadena, California 91125, USA.
authors.forename: Christine A
authors.initials: CA
authors.lastname: Romano
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Sontz
authors.forename: Jacqueline K
authors.initials: JK
authors.lastname: Barton
mesh_terms: D020137:Base Pair Mismatch / Q000235:genetics; D004249:DNA Damage / Q000235:genetics*; D045647:DNA Glycosylases / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D004260:DNA Repair / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D021381:Protein Transport / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase