Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

• Published in: Biochemical and biophysical research communications Vol. 434; no. 2; pp. 341 - 345
• Main Authors: Hata, Kuniki, Urushibara, Ayumi, Yamashita, Shinichi, Shikazono, Naoya, Yokoya, Akinari, Katsumura, Yosuke
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.05.2013
• Subjects: ANTIOXIDANTS; Antioxidants - chemistry; AQUEOUS SOLUTIONS; ASCORBIC ACID; Ascorbic Acid - chemistry; BIOLOGICAL REPAIR; Chemical repair; CONCENTRATION RATIO; DNA; DNA Breaks, Single-Stranded - drug effects; DNA Repair; DNA, Bacterial - drug effects; DNA, Bacterial - genetics; DNA, Bacterial - radiation effects; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Escherichia coli - genetics; Gamma Rays; GELS; IRRADIATION; Nucleic Acid Conformation; Plasmids - drug effects; Plasmids - genetics; Plasmids - radiation effects; PRECURSOR; Radiation damage; RADIATION PROTECTION; RADIATION, THERMAL, AND OTHER ENVIRONMENTAL POLLUTANT EFFECTS ON LIVING ORGANISMS AND BIOLOGICAL MATERIALS; Radiation-Protective Agents - chemistry; RADIOLYSIS; Solutions - metabolism; STRAND BREAKS; THERMODYNAMIC ACTIVITY; Water - metabolism; Antioxidants; Ascorbic acid; Chemical repair; Radiation damage
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2013.03.075

•We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid.•The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis.•We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites.•However, ascorbic acid seldom repairs precursors of DNA-strand breaks. We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.