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Endogenous and exogenous DNA lesions recognized by N-alkylpurine-DNA glycosylases
1Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw.
Abstract:
The combined action of glycosylases and abasic site-specific endonucleases on damaged bases in DNA results in single strand breaks. In plasmid DNA, as a consequence, the covalently closed circular (ccc) form is converted to the open circular (oc) form, and this can be quantitated by agarose gel electrophoresis. We studied DNA lesions sensitive to E. coli 3-methyladenine-DNA glycosylase II (AlkA) and cloned human N-alkylpurine-DNA glycosylase (ANPG-40) which are known to excise alkylated bases and etheno adducts. pBR322 and pAlk10 plasmids not pretreated with mutagens were cleaved by both glycosylases in the presence of enzymes possessing endonucleolytic activity, which indicates that plasmids contain unknown, endogenously formed adducts. Plasmids pretreated with chloroacetaldehyde, a mutagen forming etheno adducts, exhibited enhanced sensitivity to both glycosylases. Adducts formed by acrolein and croton aldehyde were excised by AlkA, but not by ANPG-40, whereas malondialdehyde adducts were not excised by either glycosylase. Bulky p-benzochinone adducts were not excised by AlkA, however, the plasmid pretreated with this mutagen was incised by endonucleases, possibly without prior generation of an abasic site. These examples show that examination of conformational changes of plasmid DNA can be taken advantage of to study the specificity of N-alkylpurine-DNA-glycosylases.
Insights
Researchers investigated DNA repair enzymes, specifically E. coli 3-methyladenine-DNA glycosylase II (AlkA) and human N-alkylpurine-DNA glycosylase (ANPG-40), to understand their specificity in excising DNA adducts. Plasmid DNA conformational changes were used to analyze enzyme activity and DNA lesion repair.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Enzymology
Background:
- DNA glycosylases and endonucleases create single-strand breaks in DNA by acting on damaged bases.
- Conformational changes in plasmid DNA (ccc to oc form) can be quantified via agarose gel electrophoresis to study these breaks.
- Specific DNA glycosylases, like E. coli 3-methyladenine-DNA glycosylase II (AlkA) and human N-alkylpurine-DNA glycosylase (ANPG-40), target alkylated bases and etheno adducts.
Purpose of the Study:
- To investigate the substrate specificity of AlkA and ANPG-40 towards various DNA adducts.
- To utilize plasmid DNA conformational changes as a method for assessing N-alkylpurine-DNA glycosylase activity.
- To identify unknown endogenous DNA adducts in plasmids.
Main Methods:
- Treatment of pBR322 and pAlk10 plasmids with various chemical mutagens (chloroacetaldehyde, acrolein, croton aldehyde, malondialdehyde, p-benzoquinone).
- Incubation of treated plasmids with purified E. coli AlkA and human ANPG-40 glycosylases, in the presence of endonucleases.
- Analysis of plasmid DNA conformational changes (ccc to oc conversion) using agarose gel electrophoresis to quantify DNA strand breaks.
Main Results:
- Both AlkA and ANPG-40 cleaved untreated plasmids, suggesting the presence of endogenous DNA adducts.
- Plasmids treated with chloroacetaldehyde showed increased sensitivity to both glycosylases.
- Acrolein and croton aldehyde adducts were excised by AlkA but not ANPG-40; malondialdehyde adducts were not excised by either.
- Bulky p-benzoquinone adducts were not excised by AlkA, but induced endonuclease cleavage, potentially independent of abasic sites.
Conclusions:
- Plasmid DNA conformational analysis is a viable method for studying the specificity of N-alkylpurine-DNA glycosylases.
- AlkA and ANPG-40 exhibit distinct substrate specificities for different types of DNA adducts.
- The study highlights the presence of endogenous DNA damage and the diverse repair capabilities of DNA glycosylases.