title: Single-molecule visualization of ROS-induced DNA damage in large DNA molecules.
abstract: We present a single molecule visualization approach for the quantitative analysis of reactive oxygen species (ROS) induced DNA damage, such as base oxidation and single stranded breaks in large DNA molecules. We utilized the Fenton reaction to generate DNA damage with subsequent enzymatic treatment using a mixture of three types of glycosylases to remove oxidized bases, and then fluorescent labeling on damaged lesions via nick translation. This single molecule analytical platform provided the capability to count one or two damaged sites per λ DNA molecule (48.5 kb), which were reliably dependent on the concentrations of hydrogen peroxide and ferrous ion at the micromolar level. More importantly, the labeled damaged sites that were visualized under a microscope provided positional information, which offered the capability of comparing DNA damaged sites with the in silico genomic map to reveal sequence specificity that GTGR is more sensitive to oxidative damage. Consequently, single DNA molecule analysis provides a sensitive analytical platform for ROS-induced DNA damage and suggests an interesting biochemical insight that the genome primarily active during the lysogenic cycle may have less probability for oxidative DNA damage.
authors.affiliation: Department of Chemistry and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, 121-742, Korea. jokyubong@sogang.ac.kr.
authors.forename: Jinyong
authors.initials: J
authors.lastname: Lee
authors.forename: Yongkyun
authors.initials: Y
authors.lastname: Kim
authors.forename: Sangyong
authors.initials: S
authors.lastname: Lim
authors.forename: Kyubong
authors.initials: K
authors.lastname: Jo
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics; D001583:Benzoxazoles / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D002413:Cations, Divalent; D004249:DNA Damage; D004279:DNA, Viral / Q000737:chemistry*; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D008856:Microscopy, Fluorescence; D011806:Quinolinium Compounds / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000737:chemistry*; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: C569686:Alexa Fluor 647; D001583:Benzoxazoles; D002232:Carbocyanines; D002413:Cations, Divalent; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D011806:Quinolinium Compounds; D017382:Reactive Oxygen Species; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D006861:Hydrogen Peroxide; D007501:Iron; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; C468346:Nei protein, E coli; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli