The dependence of DNase I activity on the conformation of oligodeoxynucleotides

D. H. Sutton, G. L. Conn, T. Brown, A. N. Lane

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

We have developed a sensitive continuous assay for nucleases using proton release. The assay has been applied to the determination of the kinetics of DNase I acting on short, defined deoxyoligonucleotides. The dependence of K(cat)/K(m) on sequence and structure of short oligonucleotide substrates has been measured: increasing lengths of A(n)T(n) sequences decrease the rate of cleavage. G·A mismatches in which the bases pair using imino protons are cleaved quite effectively by DNase I. In contrast, tandem G·A mismatches which use amino pairing and have B(II) phosphodiesters, are refractory to DNase I. Also, the DNA strands of DNA RNA hybrid duplexes are not cleaved by DNase I. These results show that the global conformation of a duplex and the details of its minor groove affect the cleavage efficiency by DNase I. The assay has also been used to measure the inhibition constant of the minor-groove-binding ligand propamidine. A value of 3 μM has been determined for binding to the sequence d(CGCGAATTCGCG)2, showing that dissociation constants can be determined even when there are no convenient optical signals for titrations.

Original languageEnglish
Pages (from-to)481-486
Number of pages6
JournalBiochemical Journal
Volume321
Issue number2
DOIs
StatePublished - 1997

Bibliographical note

Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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