Biomedical subjects

P C Grevatt

Publications and source records attributed to P C Grevatt.

4 recordsLinked to original sources

PubMed1992

In vitro mispairing specificity of O2-ethylthymidine.

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)

Base Sequence↗

PubMed1992

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.

Base Sequence↗

PubMed1991

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.

Alkylating Agents↗

PubMed1990

Incorporation of dA opposite N3-ethylthymidine terminates in vitro DNA synthesis.

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.

Bacteriophage phi X 174↗