Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI.

J. W. Bodnar, W. Zempsky, D. Warder, Clare M Bergson, D. C. Ward

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

The cleavage of specific DNA sequences by the restriction endonucleases AluI, DdeI, HinfI, RsaI, and TaqI has been studied by monitoring the effect of various nucleotide modifications on the rate of DNA digestion. Bacteriophage fd DNA was completely substituted in one strand with a single nucleotide analog, using an in vitro primed DNA synthesis reaction on a single-stranded viral DNA template. Twelve deoxynucleotide analogs were incorporated into these DNA substrates: 2-aminopurine, 2,6-diaminopurine, deoxytubercidin, deoxyuridine, 5-bromodeoxyuridine, 5-allylamine deoxyuridine, 5-biotinyl deoxyuridine, deoxypseudouridine, deoxyinosine, 8-azadeoxyguanosine, 5-iododeoxycytidine, and 5-bromodeoxycytidine. The restriction enzymes tested varied considerably in their ability to digest hemi-substituted DNAs containing these modified nucleotides. Structural alterations in the base pairs immediately adjacent to the phosphodiester bonds cleaved by the enzyme reduced the rate of enzyme activity most dramatically, and in most cases more than a single determinant on each base pair altered activity. Interactions with nucleotides outside the recognition site seem to have little importance in the binding or catalytic activity of these enzymes.

Original languageEnglish (US)
Pages (from-to)15206-15213
Number of pages8
JournalJournal of Biological Chemistry
Volume258
Issue number24
StatePublished - Dec 25 1983
Externally publishedYes

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DNA Restriction Enzymes
Nucleotides
Deoxyuridine
DNA
Enzymes
Base Pairing
Bromodeoxycytidine
2-Aminopurine
Allylamine
Bacteriophage M13
Bacteriophages
Single-Stranded DNA
DNA sequences
Viral DNA
Enzyme activity
Bromodeoxyuridine
Digestion
Catalyst activity
Monitoring
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI. / Bodnar, J. W.; Zempsky, W.; Warder, D.; Bergson, Clare M; Ward, D. C.

In: Journal of Biological Chemistry, Vol. 258, No. 24, 25.12.1983, p. 15206-15213.

Research output: Contribution to journalArticle

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