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Deoxyhexanucleotide containing a vinyl-chloride induced dna lesion, 1,n6-ethenoadenine - synthesis, physical characterization, and incorporation into a duplex bacteriophage-m13 genome as part of an amber codon

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Overview

authors

  • Basu, A. K.
  • Niedernhofer, Laura
  • Essigmann, J. M.

publication date

  • September 1987

journal

  • Biochemistry  Journal

abstract

  • Organic synthesis and recombinant DNA techniques have been used to situate a single 1,N6-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. Mild nonaqueous conditions were employed for deprotection because of the unstable nature of the epsilon Ade adduct in aqueous basic milieu. Physical studies involving fluorescence, circular dichroism, and 1H 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; the latter was constructed by blunt-end ligation of d(GCTAGC) in the center of the unique SmaI site of M13mp19. 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'-32P]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 adduct was introduced into a unique NheI site, and it was observed that this restriction endonuclease was able to cleave the adducted genome, albeit at a lower rate compared to unmodified DNA. 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.

subject areas

  • Adenosine
  • Base Sequence
  • Codon
  • Coliphages
  • DNA Damage
  • Escherichia coli
  • Genes, Viral
  • Indicators and Reagents
  • Magnetic Resonance Spectroscopy
  • Nucleic Acid Conformation
  • Nucleotide Mapping
  • Oligodeoxyribonucleotides
  • RNA, Messenger
  • Vinyl Chloride
  • Vinyl Compounds
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Identity

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi00392a007

PubMed ID

  • 3314993
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Additional Document Info

start page

  • 5626

end page

  • 5635

volume

  • 26

issue

  • 18

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