Abstract
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.
Highlights
The significance ofDNA ethylation at the central known for only DNA alkylation productsat the 0-6position hydrogen-bonding site (N3) of thymine was investi- of guanine (06-alkyl-dG’) and the 0 - 4 position of thymine gated using an in vitro DNA replication system
The percent extension of the primer was calculated from sis product represents no block during DNA replication with the synthesis proceeding to the 5‘-end of the 34-nucleotide template
The results demonstrate that the N3-Et-dT leto change even if the extension of the primer is inhibited at sion, present in a natural DNA template, provides a strong the initiation stage, and it is far easier to estimate the relative block to DNA replication by KfPolI with the DNA synthesis percentage of DNA synthesis products
Summary
N3 process premutagenic ENU-induced lesions to yield G - C + A. The details of the synthesis of the site-modified oligomer and its characterizationhave been described (Bhanot etal., 1990). Formation of the Site-modified Primed Template-The primed template, with the 3‘ terminus of the primer eight nucleotides away from the N3-Et-dTlesion in the templatew, as prepared by annealing is observed with N-methyl-N-nitrosourea inSOS-induced E. coli (Cuoto et al, 1989).Alkylated thymidine lesions, which normally block DNA replication, have been implicated in A. 1982).Using the natural metal ion Mg2+,the N3-Etd T present at a single site in thebacteriophage (PX174 DNA sequence blocked DNA synthesis by the Klenow fragment of E. coli polymerase I (KfPolI)(Grevatt et al, 1990). In this paper we report that in the presence of mutagenic metal ion Mn”, KfPolI incorporated dA and dTopposite the noncoding N3-Et-dT lesion present in atemplate corresponding to the PX174 DNA sequence. Cogene neu by a n A - T-+T A transversion mutation in rat neuroblastomas induced by ENU (Bargmann et al, 1986; Perantoni et al, 1987)
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