Detection of alkylation damage in human lymphocyte DNA with the comet assay.
The enzyme 3-methyladenine DNA glycosylase II (AlkA) is a bacterial repair enzyme that acts preferentially at 3-methyladenine residues in DNA, releasing the damaged base. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay (single cell gel electrophoresis) they appear as DNA strand breaks. AlkA is no t lesion-specific, but has a low activity even w ith undamagedbases. We have tested the enzyme at different concentrations to find conditions that maximise detection of alkylated bases with minimal attack on normal, undamaged DNA. AlkA detects damage in the DNA of cells treated with low concentrations of methyl methanesulphonate. We also find low background levels of alkylated bases in normal human lymphocytes.
Collins
A R
AR
Rowett Research Institute, Aberdeen, UK. a.collins@rri.sari.ac.uk
Dusinská
M
M
Horská
A
A
D000478:Alkylation; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage / Q000187:drug effects; D045647:DNA Glycosylases; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D013997:Time Factors
D008741:Methyl Methanesulfonate; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C076131:DNA-3-methyladenine glycosidase II