In vitro mispairing specificity of O2-ethylthymidine

Abstract

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)

The Role of Mutagenic Metal Ions in Mediating In Vitro Mispairing by Alkylpyrimidines

“…Disruption of normal base pairing may inhibit DNA synthesis. This is consistent with our DNA replication studies where, in the presence of the natural metal activator Mg", 02-Et-dT blocked DNA replication by Kf Pol I predominantly 3' to the lesion (41). DNA synthesis past the lesion was negligible (<1%).…”

“…This would suggest that incorporation of dA opposite O-Et-dT and extension of the resulting 02-EtdT-dA base pair will be inefficient. This is supported by in vitro DNA replication studies in the presence of Mg", where DNA synthesis was terminated predominantly (94%) 3' to 0-Et-dT, and postlesion synthesis did not occur (41). Similar results were obtained during DNA replication using bacteriophage T7 DNA polymerase (T7 Pol) (42).…”

“…The postlesion synthesis remained negligible (41). The 0--Et-dT'dA base pair may occur with the formation of two hydrogen bonds between the 04 of O-Et-dT and the N6 hydrogen atom of dA, and between the N3 of (I-Et-dT and the protonated M of dA.…”

“…DNA synthesis past the lesion was negligible (<1%). Incorporation of dA opposite oEt-dT occurred with increasing deoxyribonucleoside-5'-triphosphate (dNTP) concentrations (41), which was further enhanced by inhibiting the 3'-5' exonuclease proofreading activity of the polymerase with deoxyadenosine-5'-phosphate.…”

“…Inhibition of DNA synthesis after incorporation of dATP opposite template 0(-Et-dT (41) or 02-Et-dT-5'-triphosphate opposite template dA (66) suggests that the geometric conformation of the o2-Et-dT-dA base pair deviates significantly from that of the normal Watson-Crick pair and may adopt a wobble conformation (64,67). In the wobble conformation, phosphodiester links (both 3' and 5' to dA) may have to be distorted to accommodate the 0 -Et-dT-dA base pair in a DNA helix.…”