Deoxyhexanucleotide containing a vinyl chloride induced DNA lesion, 1,N/sup 6/-ethenoadenine: synthesis, physical characterization, and incorporation into a duplex bacteriophage M13 genome as part of an amber codon
Organic synthesis and recombinant DNA techniques have been used to situate a single 1,N/sup 6/-ethenoadenine (epsilon Ade) DNA adduct at an amber codon in the genome of an M13mp19 phage derivative. The deoxyhexanucleotide d(GCT(epsilon A)GC) was chemically synthesized by the phosphotriester method. Physical studies involving fluorescence, circular dichroism , and /sup 1/H NMR indicated epsilon Ade to be very efficiently stacked in the hexamer, especially with the 5'-thymine. Melting profile and circular dichroism studies provided evidence of the loss of base-pairing capabilities attendant with formation of the etheno ring. The modified hexanucleotide was incorporated into a six-base gap formed in the genome of an M13mp19 insertion mutant. Phage of the insertion mutant, M13mp19-NheI, produced light blue plaques on SupE strains because of the introduced amber codon. Formation of a hybrid between the single-strand DNA (plus strand) of M13mp19-NheI with SmaI-linearized M13mp19 replicative form produced a heteroduplex with a six-base gap in the minus strand. The modified hexamer (5'-/sup 32/P)d-(GCT(epsilon A)GC), after 5'-phosphorylation, was ligated into this gap by using bacteriophage T4 DNA ligase to generate a singly adducted genome with epsilon Ade at minus strand position 6274. Introduction of the radiolabel provided a useful marker for characterization of the singly adducted genome, and indeed the label appeared in the anticipated fragments when digested by several restriction endonucleases. Evidence that ligation occurred on both 5' and 3' sides of the oligonucleotide also was obtained. The M13mp19-NheI genome containing epsilon Ade will be used as a probe for studying mutagenesis and repair of this DNA adduct in Escherichia coli.
- Research Organization:
- Massachusetts Institute of Technology, Cambridge
- OSTI ID:
- 5927235
- Journal Information:
- Biochemistry; (United States), Vol. 26:18
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
62 RADIOLOGY AND NUCLEAR MEDICINE
DNA ADDUCTS
NUCLEAR MAGNETIC RESONANCE
LABELLED COMPOUNDS
BIOSYNTHESIS
NUCLEOTIDES
VINYL CHLORIDE
GENETIC EFFECTS
ATP
BACTERIOPHAGES
CODONS
DICHROISM
DNA
PHOSPHORUS 32
PROTONS
ADDUCTS
BARYONS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
CHLORINATED ALIPHATIC HYDROCARBONS
DAYS LIVING RADIOISOTOPES
ELEMENTARY PARTICLES
FERMIONS
HADRONS
HALOGENATED ALIPHATIC HYDROCARBONS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEONS
ODD-ODD NUCLEI
ORGANIC CHLORINE COMPOUNDS
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
PARASITES
PHOSPHORUS ISOTOPES
RADIOISOTOPES
RESONANCE
SYNTHESIS
VIRUSES
560300* - Chemicals Metabolism & Toxicology
550601 - Medicine- Unsealed Radionuclides in Diagnostics