Short interspersed DNA element-mediated detection of UVB-induced DNA damage and repair in the mouse genome, in vitro, and in vivo in skin.
We report a sensitive, SINE (Short Interspersed DNA Element)-mediated, PCR-based, DNA damage detection assay. Here, the SINE assay is used for detection of UVB-induced DNA damage and repair in cultured mouse cells and in vivo, in mouse skin. The unique feature of the SINE assay is its ability to support simultaneous amplification of multiple, random segments of genomic DNA. This can be accomplished due to the remarkable abundance, dispersion and conservation of SINEs in mammalian genomes. The most abundant SINEs in the mouse genome are the B1 elements, at a copy number of 50,000-80,000. Due to their strong sequence conservation, primers complementary to the B1 consensus sequence anneal to the majority of their targets in the genome. Consequently, long segments of genomic DNA located between pairs of B1 elements are efficiently amplified by PCR. Thus, in conjunction with the fact that many types of DNA adducts form blocks for thermostable polymerase, the B1 element anchored PCR makes a sensitive and versatile tool for assessing the overall integrity of the transcribed regions in mouse genome. We measured UVB-dose (0.1-3 kJ m-2) dependent formation of photoproducts in DNA from cultured cells, and after 20 h observed a substantial removal of damage at doses lower or equal to 0.6 kJ m-2. The sensitivity of detection of UVB-photoproducts formation and repair was compared to that of the conventional, single locus-targeting QPCR. Using the SINE assay we also have shown the distribution of UVB and UVC induced DNA adducts at a single nucleotide resolution within the B1 elements in mouse DNA. Lastly, we demonstrated that the sensitivity of the SINE assay is adequate for measurement of UVB-dose (1-6 kJ m-2) dependent formation and subsequent removal of photoproducts in vivo, in mouse skin.
Wang
G
G
Shriners Hospitals for Children, Department of Surgery, University of Texas Medical Branch, Galveston 77550-2725, USA.
Hallberg
L M
LM
Saphier
E
E
Englander
E W
EW
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D018736:DNA Adducts / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004307:Dose-Response Relationship, Radiation; D005260:Female; D016678:Genome; D066298:In Vitro Techniques; D051379:Mice; D008812:Mice, Hairless; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D020086:Short Interspersed Nucleotide Elements; D012867:Skin / Q000378:metabolism / Q000528:radiation effects; D014466:Ultraviolet Rays / Q000009:adverse effects*
D018736:DNA Adducts; D017931:DNA Primers