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Use of a dodecadeoxynucleotide to study repair of the O4-methylthymine lesion
M E Dolan1, M Oplinger, A E Pegg
1Department of Physiology, Milton S. Hershey Medical Center, PA 17033.
Abstract:
A dodecadeoxynucleotide of defined sequence containing O4-methylthymine was labeled at the 5' end with [32P] by the reaction with (gamma-32P]ATP and polynucleotide kinase. Extracts prepared from bacterial and mammalian sources such as the human cell lines, HeLa and HT29, and rat liver were incubated with the labeled, methylated dodecamer to determine the extent of repair of the lesion. The labeled, demethylated dodecamer was separated from the labeled methylated dodecamer on a reverse-phase column using a shallow methanol gradient. There was complete repair of O4-methylthymine by the E. coli alkyltransferase upon incubation for 4 h at 37 degrees C. There was no detectable amount of demethylated product formed upon incubation with HeLa or HT29 cell extract for the same incubation period. There was also no repair of the O4-methylthymine lesion in the presence of crude rat-liver extract. However, the rat-liver extract alone degraded the methylated substrate completely, and the assay had to be conducted in the presence of NaF, AMP and unlabeled, nonmethylated dodecamer to prevent this. The results obtained from this assay, which is at least an order of magnitude more sensitive than previous methods, are in agreement with previous results that the mammalian alkyltransferase is specific for O6-alkylguanine repair.
Insights
Bacterial E. coli alkyltransferase fully repaired O4-methylthymine lesions. Mammalian cell extracts from HeLa, HT29, and rat liver showed no repair, suggesting mammalian alkyltransferases are specific for O6-alkylguanine.
Area of Science:
- Molecular Biology
- Biochemistry
- DNA Repair Mechanisms
Background:
- DNA damage, including alkylation, poses a threat to genomic integrity.
- O4-methylthymine is a DNA lesion that can arise from exposure to alkylating agents.
- DNA repair pathways are crucial for maintaining cellular health and preventing mutations.
Purpose of the Study:
- To investigate the repair capacity of bacterial and mammalian extracts for O4-methylthymine DNA lesions.
- To determine if mammalian alkyltransferases, known for O6-alkylguanine repair, can also repair O4-methylthymine.
- To develop a sensitive assay for detecting DNA repair of methylated thymine.
Main Methods:
- Synthesis of a 5'-[32P]-labeled dodecadeoxynucleotide containing O4-methylthymine.
- Incubation of the labeled substrate with bacterial (E. coli) and mammalian (HeLa, HT29, rat liver) cell extracts.
- Separation of repaired (demethylated) and unrepaired (methylated) DNA using reverse-phase HPLC.
- Quantification of repair by measuring the conversion of the methylated lesion.
Main Results:
- E. coli alkyltransferase completely repaired the O4-methylthymine lesion within 4 hours at 37°C.
- No detectable repair of O4-methylthymine was observed with HeLa or HT29 cell extracts.
- Crude rat liver extract degraded the substrate, but specific repair was absent.
- The assay demonstrated high sensitivity, exceeding previous methods by an order of magnitude.
Conclusions:
- Bacterial E. coli alkyltransferase efficiently repairs O4-methylthymine.
- Mammalian alkyltransferases, including those in human and rat liver cells, do not appear to repair O4-methylthymine.
- These findings support the hypothesis that mammalian alkyltransferases are primarily specific for O6-alkylguanine repair, not O4-methylthymine.