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.

The O2-position of thymine is a major site of base alkylation by N-nitroso-alkylating agents, and its biological relevance remains obscure. The potential significance of this DNA damage was ascertained by studying in vitro DNA replication properties of O2-ethylthymidine (O2-Et-dT) site-specifically incorporated into a 36-nucleotide template. DNA replication was initiated eight nucleotides away from the O2-Et-dT lesion by Escherichia coli polymerase I (Klenow fragment) using a 17-nucleotide primer. In the presence of 10 microM dNTP and Mg2+, O2-Et-dT blocked DNA replication predominantly (94%) 3' to O2-Et-dT, with the remainder (5%) blocked after incorporation of a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product). Postlesion synthesis was negligible (less than 1%). Nucleotide incorporation opposite O2-Et-dT increased to 23% at 200 microM dNTP. Postlesion synthesis remained negligible (less than 2%). DNA sequencing revealed dA present opposite O2-Et-dT in the incorporation-dependent blocked product. Negligible postlesion synthesis suggests that incorporation of dA opposite O2-Et-dT inhibits in vitro DNA synthesis. The O2-Et-dT.dA base pair may also impede DNA synthesis in vivo, contributing to the cytotoxicity of the ethylating agents. Substitution of Mn2+ for Mg2+ enhanced nucleotide incorporation opposite O2-Et-dT and produced postlesion synthesis (16%) at 10 microM dNTP, which increased to 39% at 200 microM dNTP. DNA sequence analysis showed that while dA was present opposite O2-Et-dT in the incorporation-dependent blocked product, both dA and dT were present opposite this lesion in the postlesion synthesis product.(ABSTRACT TRUNCATED AT 250 WORDS)

A bacteriophage 4X174-based site-specific mutagenesis system for the study of the in vivo mutagenic frequency and specificity of carcinogen-induced modification in DNA is presented. A (-)-strand primer containing 06 methylguanine in a specific site was hybridized to a singlestranded region in gene G of OX gapped duplex DNA. The hybrid was enzymatically converted to replicative form DNA and was used to transform Escherichia coli cells. All gene G mutants generated by the modification were rescued by genetic complementation. An amber mutation in lysis gene E of the (+) strand of the replicative form DNA prevented lytic growth of wild-type phage derived from this strand. In each mutantcontaining infective center produced from the transformed cells, gene G mutant phage were present in a 3:1 ratio compared to wild type. Thus, in vivo, 06-methylguanine in replicating OX DNA has a mutagenic frequency of 75%. When repair of 06 methylguanine occurred, it was prereplicative. The mutations were due exclusively to the misincorporation of thymine.Most chemical carcinogens are mutagens (1) that react with DNA to form various covalent adducts (2, 3). To establish a cause-and-effect relationship between the specific DNA lesions and the specific mutations produced, we developed a system for assaying site-specific mutagenesis in gene G of bacteriophage 4X174 (4, 5). Gene G codes for the phage spike protein, which is essential for the formation of infectious phage. The system produced mutant phage at low percentage yield (4) due to large numbers of(+)-strand-derived wild-type (wt) phage. This also distorts the true percentage of any wt phage produced from the adduct-containing (-) strand. Low mutant recovery was a general problem in the study of site-specific mutagenesis, and strategies have been developed in different systems to increase mutant yields (6-10).We here describe a version of the 4X174-based mutagenesis system. A complementation plasmid efficiently supports growth of gene G mutants, and an amber (am) mutation in the lysis gene E of the (+) strand of the transforming OX

N3-Ethylthymidine (N3-Et-dT) was site specifically incorporated into a 17-nucleotide oligomer to investigate the significance of DNA ethylation at the central hydrogen-bonding site (N3) of thymine. The 5'-(dimethoxytrityl)-protected N3-Et-dT was converted to the corresponding 3'-phosphoramidite and used to incorporate N3-Et-dT at a single site in the oligonucleotide during synthesis by the phosphite triester method. The purified N3-Et-dT-containing oligomer was ligated to a second 17-mer to yield a 34-nucleotide template with N3-Et-dT present at position 26 from the 3'-end. The template DNA, which corresponds to a specific sequence at gene G of bacteriophage phi X174, was used to study the specificity of nucleotide incorporation opposite N3-Et-dT. At 10 microM dNTP and 5 mM Mg2+, N3-Et-dT blocked DNA synthesis by Escherichia coli polymerase I (Klenow fragment): 96% immediately 3' to N3-Et-dT and 4% after incorporation of a nucleotide opposite N3-Et-dT (incorporation-dependent blocked product). DNA replication past the lesion (postlesion synthesis) was negligible. Incorporation opposite N3-Et-dT increased with increased dNTP concentrations, reaching 35% at 200 microM. Postlesion synthesis remained negligible. DNA sequencing of the incorporation-dependent blocked product revealed that dA is incorporated opposite N3-Et-dT consistent with the "A" rule in mutagenesis. Formation of the N3-Et-dT.dA base pair at the 3'-end of the growing chain terminated DNA synthesis. These results implicate N3-Et-dT as a potentially cytotoxic lesion produced by ethylating agents.