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Hydrolysis of carcinogen--DNA adducts by three classes of deoxyribonucleosidase to their corresponding bases
1Division of Environmental Science, Columbia University, New York, NY 10032.
Abstract:
We have investigated three different enzymes, each of which splits the glycosidic bonds of normal deoxyribonucleosides, for their ability to cleave acid-sensitive carcinogen-DNA adducts: Leishmania donovani purine deoxyribonucleosidase; Lactobacillus helveticus trans-N-deoxyribosylase; and purine nucleoside phosphorylase. Twenty-eight substrates were assayed using one or more of the enzymes, monitoring free base formation by reverse-phase HPLC and a diode array detector. Products were characterized by their retention times and UV spectra. The deoxyribonucleosidase showed the broadest specificity: substrates cleaved by either the trans-N-deoxyribosylase or the phosphorylase were hydrolyzed by the deoxyribonucleosidase. O6- and N2-alkyldeoxyguanosine, N6-alkyl deoxyadenosine and several cyclic adducts were all hydrolyzed by the deoxyribonucleosidase. None of these enzymes cleaved three bulkier adducts; N2-, N6- and C-8 derivatives of benzo[a]pyrene, benzo[c]phenanthrene and N-acetoxy-2-acetylaminofluorene, respectively. Only the deoxyribonucleosidase hydrolyzed O6-methyldeoxyguanosine while both the deoxyribonucleosidase and trans-N-deoxyribosylase cleaved N6-methyl deoxyadenosine. These enzymes, especially the deoxyribonucleosidase, provide mild ways to release the carcinogen-modified base from deoxyribose and will be useful in DNA adducts analysis.
Insights
Three enzymes were tested for cleaving carcinogen-DNA adducts. Leishmania donovani purine deoxyribonucleosidase showed broad specificity, effectively releasing modified bases from deoxyribose for DNA adduct analysis.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Carcinogen-DNA adducts are critical biomarkers of exposure and can lead to mutations.
- Mild and specific methods are needed for analyzing these adducts.
Purpose of the Study:
- To evaluate three enzymes for their ability to cleave glycosidic bonds in carcinogen-DNA adducts.
- To determine the substrate specificity of these enzymes against various modified deoxyribonucleosides.
Main Methods:
- Assayed 28 deoxyribonucleoside substrates using Leishmania donovani purine deoxyribonucleosidase, Lactobacillus helveticus trans-N-deoxyribosylase, and purine nucleoside phosphorylase.
- Monitored free base formation via reverse-phase HPLC with a diode array detector.
- Characterized products by retention times and UV spectra.
Main Results:
- Leishmania donovani purine deoxyribonucleosidase exhibited the broadest specificity, hydrolyzing substrates cleaved by the other two enzymes.
- This deoxyribonucleosidase cleaved O6- and N2-alkyldeoxyguanosine, N6-alkyl deoxyadenosine, and cyclic adducts.
- None of the enzymes cleaved bulky polycyclic aromatic hydrocarbon or acetylaminofluorene derivatives; only deoxyribonucleosidase cleaved O6-methyldeoxyguanosine.
Conclusions:
- Leishmania donovani purine deoxyribonucleosidase is a versatile enzyme for cleaving various carcinogen-DNA adducts.
- These enzymes offer mild and effective methods for releasing modified bases from deoxyribose, aiding in DNA adduct analysis.