Mutants of the base excision repair glycosylase, endonuclease III: DNA charge transport as a first step in lesion detection.
Biochemistry
; 50(27): 6133-45, 2011 Jul 12.
Article
in En
| MEDLINE
| ID: mdl-21651304
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA Damage
/
DNA, Bacterial
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Mutagenesis, Site-Directed
/
Escherichia coli Proteins
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Deoxyribonuclease (Pyrimidine Dimer)
/
DNA Repair
/
Escherichia coli
Type of study:
Diagnostic_studies
Language:
En
Journal:
Biochemistry
Year:
2011
Document type:
Article
Affiliation country: