The role of N3-ethyldeoxythymidine in mutagenesis and cytotoxicity by ethylating agents.
The significance of DNA ethylation at the central hydrogen-bonding site (N3) of thymine was investigated using an in vitro DNA replication system. The system utilized a primed template in which the 3'-end of the primer is eight nucleotides away from N3-ethyldeoxythymidine (N3-Et-dT), present at template position 26 from the 3'-end. The 34-nucleotide template corresponds to a specific DNA sequence at gene G of bacteriophage phi X174. DNA synthesis products were quantitated by electrophoretic separation and autoradiography. At 10 microM dNTP and 0.5 mM Mn2+, N3-Et-dT blocked DNA synthesis by Escherichia coli polymerase I (Klenow fragment): 60% after incorporating a nucleotide opposite N3-Et-dT (incorporation-dependent blocked product) and 39% 3' to N3-Et-dT. DNA replication past the lesion (post-lesion synthesis) was negligible. Post-lesion synthesis increased using higher concentrations of dNTP, reaching 68% at 200 microM dNTP. DNA sequencing revealed that dA was incorporated opposite N3-Et-dT in the incorporation-dependent blocked product. In the post-lesion synthesis product, dT was exclusively incorporated opposite N3-Et-dT. Formation of the N3-Et-dT.dA base pair at the replication fork terminated DNA synthesis, while the N3-Et-dT.dT base pair formed at the 3'-end of the growing chain was extended, leading to an A.T----T.A transversion mutation. The results suggest a dual role for the N3-Et-dT lesion, contributing in part to the cytotoxicity and mutagenicity of ethylating agents. These studies provide a basis for understanding the activation of oncogene neu by A.T----T.A transversion mutation in rat neuroblastomas induced by N-ethyl-N-nitrosourea.
Grevatt
P C
PC
Department of Environmental Medicine, New York University Medical Center, New York 10016.
Donahue
J M
JM
Bhanot
O S
OS
D000477:Alkylating Agents / Q000633:toxicity*; D000818:Animals; D001483:Base Sequence; D002470:Cell Survival / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects; D020149:Manganese Poisoning; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D051381:Rats; D013698:Templates, Genetic; D013936:Thymidine / Q000031:analogs & derivatives* / Q000633:toxicity; D014407:Tumor Cells, Cultured
D000477:Alkylating Agents; C066706:N(3)-ethylthymidine; D013936:Thymidine