Mapping oxidative DNA damage at nucleotide level

DNA damage induced by reactive oxygen species (ROS) is considered an import ant intermediate in the pathogenesis of human conditions such as cancer and aging. By developing an oxidative-induced DNA damage mapping version of th e Ligation-mediated polymerase chain reaction (LMPCR) technique, we investi gated the in vivo and in vitro frequencies of DNA base modifications caused by ROS in the human p53 and PGK1 gene. Intact human male fibroblasts were exposed to 50 mM H2O2, or purified genomic DNA was treated with 5 mM H2O2, 100 mu M Ascorbate, and 50 mu M, 100 mu M, or 100 mu M of Cu(II), Fe(III), or Cr(VI) respectively. The damage pattern generated in vivo was nearly ide ntical to the in vitro Cu(II) or Fe(III) damage patterns; damage was non-ra ndom with guanine bases heavily damaged. Cr(VI) generated an in vitro damag e pattern similar to the other metal ions, although several unique thymine positions were damaged. Also, extra nuclear sites are a major contributor o f metal ions (or metal-like ligands). These data show that the local probab ility of H2O2-mediated DNA damage is determined by the primary DNA sequence , with chromatin structure having a limited effect. The data suggest a mode l in which DNA-metal ion binding domains can accommodate different metal-io ns. LMPCR's unique aspect is a blunt-end ligation of an asymmetric double-s tranded linker, permitting exponential PCR amplification. An important fact or limiting the sensitivity of LMPCR is the representation of target gene D NA relative to non-targeted genes; therefore, we recently developed a metho d to eliminate excess non-targeted genomic DNA. Restriction enzyme-digested genomic DNA is size fractionated by Continuous Elution Electrophoresis (CE E), capturing the target sequence of interest. The amount of target DNA in the starting material for LMPCR is enriched, resulting in a stronger amplif ication signal. CEE provided a 24-fold increase in the signal strength attr ibutable to strand breaks plus modified bases created by ROS in the human p 53 and PGK1 genes, detected by LMPCR. We are currently taking advantage of the enhanced sensitivity of target gene-enriched LMPCR to map DNA damage in duced in human breast epithelial cells exposed to non-cytotoxic concentrati ons of H2O2.