Quantification of UVR-induced DNA damage: global- versus gene-specific levels of thymine dimers.
The induction and repair of DNA damage has been shown to occur heterogeneously throughout the mammalian genome. As a consequence, analysis of these parameters at a global genome level may not reflect important gene-level events. Few techniques have been established to explore quantitatively gene-specific DNA damage and repair. Most of these are polymerase chain reaction (PCR)-based assays and are relatively insensitive, relying on decreased PCR amplification arising from damage in template DNA. We have developed a quantitative assay that combines specific immunocapture of damaged DNA by an antiserum specific for thymine dimers (IgG479), with PCR amplification of a 149 bp fragment of the human H-ras proto-oncogene. Quantification of DNA damage was based upon proportionality between the amount of the PCR product and the initial amount of damage. Detection of thymine dimers was possible with nanogram amounts of genomic DNA and increased in a linear, dose-responsive manner. Using this assay, gene-level induction of thymine dimers was shown to be directly proportional to levels induced in the global genome of ultraviolet radiation (UVR)-exposed, extracted DNA as measured by gas chromatography-mass spectrometry (GC-MS). This result suggests that global damage assessments do indeed reflect gene-level events although we predict that this relationship may not be maintained when applied to a cellular system. These findings demonstrate the suitability of this approach to the detection of UVR-induced DNA damage at the level of individual genes.
Karakoula
Aikaterini
A
Oxidative Stress Group, Department of Clinical Biochemistry, University of Leicester, RKCSB, Leicester Royal Infirmary, University Hospitals of Leicester NHS Trust, PO Box 65, LE2 7LX, Leicester, UK.
Evans
Mark D
MD
Podmore
Ian D
ID
Hutchinson
Peter E
PE
Lunec
Joseph
J
Cooke
Marcus S
MS
D000918:Antibody Specificity; D004249:DNA Damage / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D011905:Genes, ras / Q000235:genetics / Q000276:immunology / Q000528:radiation effects; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D011071:Poly T / Q000276:immunology / Q000528:radiation effects; D016133:Polymerase Chain Reaction / Q000379:methods*; D000090063:Proto-Oncogene Mas; D011740:Pyrimidine Dimers / Q000032:analysis* / Q000235:genetics; D014466:Ultraviolet Rays / Q000009:adverse effects*
D007074:Immunoglobulin G; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011740:Pyrimidine Dimers; D011071:Poly T