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 Citations (Scopus)

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

Fingerprint

DNA Restriction Enzymes
Manganese
Binding Sites
Ions
DNA
Metals
Enzymes
Sugar Phosphates
DNA Cleavage
Phosphates

Keywords

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

ASJC Scopus subject areas

  • Virology

Cite this

Catalytic efficiency and sequence selectivity of a restriction endonuclease modulated by a distal manganese ion binding site. / Sam, My D.; Horton, Nancy C; Nissan, T. Amar; Perona, John J.

In: Journal of Molecular Biology, Vol. 306, No. 4, 02.03.2001, p. 851-861.

Research output: Contribution to journalArticle

@article{be0331c3472d4168bd1727c94486d9ff,
title = "Catalytic efficiency and sequence selectivity of a restriction endonuclease modulated by a distal manganese ion binding site",
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 {\AA} 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.",
keywords = "Indirect readout, Metal ion catalysis, Protein engineering, Protein-nucleic acid recognition",
author = "Sam, {My D.} and Horton, {Nancy C} and Nissan, {T. Amar} and Perona, {John J.}",
year = "2001",
month = "3",
day = "2",
doi = "10.1006/jmbi.2000.4434",
language = "English (US)",
volume = "306",
pages = "851--861",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "4",

}

TY - JOUR

T1 - Catalytic efficiency and sequence selectivity of a restriction endonuclease modulated by a distal manganese ion binding site

AU - Sam, My D.

AU - Horton, Nancy C

AU - Nissan, T. Amar

AU - Perona, John J.

PY - 2001/3/2

Y1 - 2001/3/2

N2 - 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.

AB - 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.

KW - Indirect readout

KW - Metal ion catalysis

KW - Protein engineering

KW - Protein-nucleic acid recognition

UR - http://www.scopus.com/inward/record.url?scp=0035793706&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035793706&partnerID=8YFLogxK

U2 - 10.1006/jmbi.2000.4434

DO - 10.1006/jmbi.2000.4434

M3 - Article

C2 - 11243793

AN - SCOPUS:0035793706

VL - 306

SP - 851

EP - 861

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 4

ER -