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Alterations in DNA-restriction enzyme interactions by O4-alkyldeoxythymidines
F C Richardson1, K K Richardson
1Toxicology Division, Eli Lilly and Co., Greenfield, Indiana 46140.
Abstract:
O4-Alkyldeoxythymidines have been extensively studied for their ability to cause mutations and to induce cancer. Since these adducts can change DNA conformation, they may also have a more immediate effect of altering DNA-protein interactions. To address this issue, the effects of these adducts on restriction enzyme activity were examined. Oligodeoxyribonucleosides containing O4-ethyldeoxythymidine (O4-EtdT) or O4-methyldeoxythymidine (O4-MedT) at a unique site within the sequence 5'-GAATGGATCCTAATGAGATC-3' were constructed by automated DNA synthesis. This sequence contains the recognition site for various restriction enzymes. These oligomers were annealed to various complementary strands and digested with restriction enzymes: BamHI or BstI (GGATCC); Sau3A, NdeII, or MboI (GATC); DpnI (GmATC); and BstYI, MflI, or XhoII (PuGATCPy). Analysis of the digests demonstrated that the presence of either O4-EtdT or O4-MedT abolished the ability of XhoII, MboI, MflI, or NdeII to cut at the restriction site. DpnI failed to cut any of the oligomers. BamHI, Sau3A, BstI, and BstYI exhibited alterations in cutting specificity depending upon the oligomers used. These results demonstrated that O4-alkyldeoxythymidine adducts alter DNA-restriction enzyme interactions in a protein- and sequence-dependent manner. Because of the importance of natural methylation in genetic regulation, it is possible that aberrant methylation in the form of DNA adducts could also alter protein-DNA interactions in cells exposed to DNA-modifying agents.
Insights
DNA damage from O4-alkyldeoxythymidine adducts disrupts DNA-protein interactions. These modified thymidines alter restriction enzyme activity, impacting DNA recognition and cutting, with implications for cellular processes.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- O4-alkyldeoxythymidines are known mutagens and carcinogens.
- These DNA adducts can alter DNA conformation, potentially affecting DNA-protein interactions.
- The impact of these adducts on specific DNA-protein interactions, like restriction enzyme activity, requires investigation.
Purpose of the Study:
- To investigate the effects of O4-ethyldeoxythymidine (O4-EtdT) and O4-methyldeoxythymidine (O4-MedT) adducts on restriction enzyme activity.
- To determine if these adducts alter the sequence-specific DNA binding and cleavage by restriction enzymes.
- To understand the implications of these alterations for DNA-protein interactions in cells exposed to DNA-modifying agents.
Main Methods:
- Automated DNA synthesis was used to create oligodeoxyribonucleosides containing O4-EtdT or O4-MedT at a specific site.
- These modified oligomers were annealed to complementary strands.
- Digestion with various restriction enzymes (BamHI, BstI, Sau3A, NdeII, MboI, DpnI, BstYI, MflI, XhoII) was analyzed.
Main Results:
- O4-EtdT and O4-MedT adducts abolished the cutting activity of XhoII, MboI, MflI, and NdeII.
- DpnI enzyme did not cut any of the modified oligomers.
- BamHI, Sau3A, BstI, and BstYI showed altered cutting specificities depending on the DNA adduct and sequence context.
Conclusions:
- O4-alkyldeoxythymidine adducts significantly alter DNA-restriction enzyme interactions.
- These alterations are dependent on the specific enzyme and DNA sequence.
- Aberrant DNA methylation, such as these adducts, can disrupt critical protein-DNA interactions within cells.