Monitoring repair of DNA damage in cell lines and human peripheral blood mononuclear cells.
We introduce a method to follow DNA repair that is suitable for both clinical and laboratory samples. An episomal construct with a unique 8-oxoguanine (8-oxoG) base at a defined position was prepared in vitro using single-stranded phage harboring a 678-bp tract from exons 5 to 9 of the human P53 gene. Mixing curve experiments showed that the real-time PCR method has a linear response to damage, suggesting that it is useful for DNA repair studies. The episomal construct with a unique 8-oxoG base was introduced into AD293 cells or human peripheral blood mononuclear cells, and plasmids were recovered as a function of time. The quantitative real-time PCR assay demonstrated that repair of the 8-oxoG was 80% complete in less than 48 h in AD293 cells. Transfection of small interfering RNAs down-regulated OGG1 expression in AD293 cells and reduced the repair of 8-oxoG to 30%. Transfection of the episome into unstimulated white blood cells showed that 8-oxoG repair had a half-life of 2 to 5h. This method is a rapid, reproducible, and robust way to monitor repair of specific adducts in virtually any cell type.
Lee
Hyun-Wook
HW
Department of Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
Lee
Hae-Jung
HJ
Hong
Chong-mu
CM
Baker
David J
DJ
Bhatia
Ravi
R
O'Connor
Timothy R
TR
D001483:Base Sequence; D002460:Cell Line; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000502:physiology*; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism