In vitro DNA replication implicates O2-ethyldeoxythymidine in transversion mutagenesis by ethylating agents.
A 36-nucleotide oligomer containing a single O2-ethyldeoxythymidine (O2-Et-dT) adduct at a specific site was synthesized. The oligomer, which corresponds to a specific DNA sequence in gene G of bacteriophage phi X174, was used as a template by T7 DNA polymerase to investigate the in vitro mutagenic specificity of O2-Et-dT. At 10 microM dNTP and 5 mM Mg++, the progress of T7 DNA polymerase was interrupted by O2-Et-dT: 80% 3' to O2-Et-dT and 14% after incorporating a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product). DNA synthesis past the lesion was low (6%). Incorporation of a nucleotide opposite O2-Et-dT and subsequent postlesion synthesis were enhanced by increasing the dNTP concentration, with postlesion synthesis reaching 30% at 200 microM. Postlesion synthesis was further increased to 45% by addition of 10 mM dAMP to the polymerization reactions. DNA sequencing revealed that both dA and dT were incorporated opposite O2-Et-dT with dA incorporation impeding the progress of DNA synthesis. dT incorporation was efficiently extended implicating O2-Et-dT in transversion mutagenesis in vivo. These studies provide a basis for understanding the molecular mechanisms by which ethylating agents contribute to cytotoxicity, A.T transversion mutagenesis and activation of the oncogene neu by an A.T----T.A transversion event in rat neuroblastomas.
Bhanot
O S
OS
Department of Environmental Medicine, New York University Medical Center, NY 10016.
Grevatt
P C
PC
Donahue
J M
JM
Gabrielides
C N
CN
Solomon
J J
JJ
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009153:Mutagens / Q000633:toxicity*; D013936:Thymidine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism
D009153:Mutagens; C019377:O(2)-ethylthymidine; D004247:DNA; D013936:Thymidine