Monitoring repair of DNA damage in cell lines and human peripheral blood mononuclear cells

Academic Article

Abstract

  • 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 5 h. This method is a rapid, reproducible, and robust way to monitor repair of specific adducts in virtually any cell type. © 2007 Elsevier Inc. All rights reserved.
  • Published In

    Digital Object Identifier (doi)

    Author List

  • Lee HW; Lee HJ; Hong CM; Baker DJ; Bhatia R; O'Connor TR
  • Start Page

  • 246
  • End Page

  • 259
  • Volume

  • 365
  • Issue

  • 2