Protective Effect of Vitamin C against Double-Strand Breaks in Reconstituted Chromatin Visualized by Single-Molecule Observation

ABSTRACT Direct attack to genomic DNA by reactive oxygen species causes various types of lesions, including base modifications and strand breaks. The most significant lesion is considered to be an unrepaired double-strand break that can lead to fatal cell damage. We directly observed double-strand breaks of DNA in reconstituted chromatin stained by a fluorescent cyanine dye, YOYO (quinolinium, 1,19-[1,3- propanediylbis[(dimethyliminio)-3,1- propanediyl]]bis[4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]]-, tetraiodide), in solution, where YOYO is known to have the ability to photo-cleave DNAs by generating reactive oxygen species. Reconstituted chromatin was assembled from large circular DNA (106 kbp) with core histone proteins. We also investigated the effect of vitamin C (ascorbic acid) on preventing photo-induced double-strand breaks in a quantitative manner. We found that DNA is protected against double-strand breaks by the addition of ascorbic acid, and this protective effect is dose dependent. The effective kinetic constant of the breakage reaction in the presence of 5 mM ascorbic acid is 20 times lower than that in the absence of ascorbic acid. This protective effect of ascorbic acid in reconstituted chromatin is discussed in relation to the highly compacted polynucleosomal structure. The results highlight the fact that single-molecule observation is a useful tool for studying double-strand breaks in giant DNA and chromatin. INTRODUCTON It is widely accepted that oxidative damage to genomic DNA by reactive oxygen species induces various types of DN