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

Journal Article · Thu Nov 01 00:00:00 EST 1990 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00497a010· OSTI ID:5475798

Bhanot, O S; Grevatt, P C; Donahue, J M; Gabrielides, C N; Solomon, J J ^[1]

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1. New York Univ. Medical Center (USA)

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 phospite 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 bacteriophase {var phi}X174, was used to study the specificity of nucleotide incorporation opposite N3-Et-dT. At 10 {mu}M dNTP and 5 mM Mg{sup 2{plus}}, N3-Et-dT blocked DNA synthesis by Escherichia coli polymerase I (Klenow fragment): 96{percent} immediately 3' to N3-Et-dT and 4{percent} 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{percent} at 200 {mu}M. 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{center dot}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.

• OSTI ID: 5475798
• Journal Information: Biochemistry; (United States), Vol. 29:45; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA
ALKYLATION
DNA REPLICATION
MOLECULAR BIOLOGY
NITROSO COMPOUNDS
DERIVATIZATION
DNA SEQUENCING
MUTAGENESIS
OLIGONUCLEOTIDES
PHOSPHORUS 32
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ISOTOPES
LIGHT NUCLEI
NUCLEI
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS ISOTOPES
RADIOISOTOPES
STRUCTURAL CHEMICAL ANALYSIS
550201* - Biochemistry- Tracer Techniques