Catalytic efficiency and sequence selectivity of a restriction endonuclease modulated by a distal manganese ion binding site

My D. Sam, Nancy C. Horton, T. Amar Nissan, John J. Perona

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

Crystal structures of EcoRV endonuclease bound in a ternary complex with cognate duplex DNA and manganese ions have previously revealed an Mn2+-binding site located between the enzyme and the DNA outside of the dyad-symmetric GATATC recognition sequence. In each of the two enzyme subunits, this metal ion bridges between a distal phosphate group of the DNA and the imidazole ring of His71. The new metal-binding site is specific to Mn2+ and is not occupied in ternary cocrystal structures with either Mg2+ or Ca2+. Characterization of the H71A and H71Q mutants of EcoRV now demonstrates that these distal Mn2+ sites significantly modulate activity toward both cognate and non-cognate DNA substrates. Single-turnover and steady-state kinetic analyses show that removal of the distal site in the mutant enzymes increases Mn2+-dependent cleavage rates of specific substrates by tenfold. Conversely, the enhancement of non-cognate cleavage at GTTATC sequences by Mn2+ is significantly attenuated in the mutants. As a consequence, under Mn2+ conditions EcoRV-H71A and EcoRV-H71Q are 100 to 700-fold more specific than the wild-type enzyme for cognate DNA relative to the GTTATC non-cognate site. These data reveal a strong dependence of DNA cleavage efficiency upon metal ion-mediated interactions located some 20 Å distant from the scissile phosphodiester linkages. They also show that discrimination of cognate versus non-cognate DNA sequences by EcoRV depends in part on contacts with the sugar-phosphate backbone outside of the target site.

Original languageEnglish (US)
Pages (from-to)851-861
Number of pages11
JournalJournal of Molecular Biology
Volume306
Issue number4
DOIs
StatePublished - Mar 2 2001
Externally publishedYes

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Keywords

  • Indirect readout
  • Metal ion catalysis
  • Protein engineering
  • Protein-nucleic acid recognition

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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