Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction
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H Rodriguez1, R Drouin, G P Holmquist
1Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, California 91010, USA.
Abstract:
The ligation-mediated polymerase chain reaction was used to map the frequency of reactive oxygen species-induced DNA damage at nucleotide resolution in genomic DNA purified from cultured human male fibroblasts. Damaged pyrimidine and purine bases were recognized and cleaved by the Nth and Fpg proteins from Escherichia coli, respectively. Strand breaks and modified bases were induced in vitro by copper ion-mediated reduction of hydrogen peroxide in the presence of ascorbate; reactant concentrations were adjusted to induce lesions at a frequency of 1 per 2-3 kilobases in purified genomic DNA. Glyoxal gel analysis demonstrated that the ratio of induced strand breaks to induced base damage was 0.8/2.7 in DNA dialyzed extensively to remove adventitious transition metal ions. Ligation-mediated polymerase chain reaction analysis of the damage frequency in the promoter region of the transcriptionally active phosphoglycerate kinase (PGK 1) gene revealed that (Cu(II)/ascorbate/H2O2 caused DNA base damage by a sequence-dependent mechanism, with the 5' bases of d(pGn) and d(pCn) being damage hot spots, as were the most internal guanines of d(pGGGCCC) and d(pCCCGGG). Since base damage occurs after formation of a DNA-Cu(I)-H2O2 complex, these data suggest that the local DNA sequence affects formation of DNA-Cu(I)-H2O2 complexes and/or the efficiency of base oxidation during resolution of this complex.
Insights
Reactive oxygen species cause DNA damage, with specific DNA sequences acting as hotspots for this damage. This finding is crucial for understanding DNA repair mechanisms and genetic mutation risks.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Reactive oxygen species (ROS) are implicated in DNA damage, contributing to aging and disease.
- Understanding the precise locations and mechanisms of ROS-induced DNA lesions is critical.
Purpose of the Study:
- To map the frequency of ROS-induced DNA damage at nucleotide resolution.
- To investigate the sequence-dependent mechanisms of DNA damage.
- To elucidate the role of DNA sequence in ROS-mediated oxidative damage.
Main Methods:
- Ligation-mediated polymerase chain reaction (LM-PCR) for nucleotide-resolution mapping.
- Enzymatic cleavage of damaged bases by E. coli Nth and Fpg proteins.
- In vitro induction of DNA lesions using copper ions, hydrogen peroxide, and ascorbate.
Main Results:
- ROS-induced DNA base damage occurred via a sequence-dependent mechanism.
- Specific sequences, including 5' d(pGn) and d(pCn), and internal guanines in G/C-rich regions, were identified as damage hotspots.
- The ratio of strand breaks to base damage was determined under specific conditions.
Conclusions:
- Local DNA sequence influences the formation of DNA-copper complexes and/or the efficiency of base oxidation.
- These findings provide insights into the sequence-context of oxidative DNA damage.
- The study highlights the importance of DNA sequence in mediating ROS-induced genotoxicity.