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Proc. Nat. Acad. Sci. USA
Vol. 72, No. 9, pp. 3310-314, September 1975
Biochemistry

Specificity of substrate recognition by the EcoRI restriction
endonuclease
(P)NA/plasmid/simian virus 40/modification)

BARRY POLISKY*, PATRICIA GREENEt, DAVID E. GARFINt, BRIAN J. MCCARTHY*, HOWARD M.
GOODMAN*, AND HERBERT W. BOYERt
* Department of Biochemistry and Biophysics and t Department ofMicrobiology, University of California, San Francisco, San Francisco, Calif. 94143

Communicated by Norman Davidson, May 23,1975

ABSTRACT The substrate specificity of the EcoRI restriction endonuclease can be varied in vitro by changing the
pH and the ionic environment of the reaction. Phosphodiester bond cleavage occurs at a DNA hexanucleotide sed(N-G-A-A-T-T-C-N)
quence
quned(N-C-T-T-A-A-G-N)when the ionic strength is high,
100 mM Tris'HCI, 50 mM NaCl, 5 mM MgCl2, and the pH is
approximately 7.3. Lowering the ionic strength to 25 mM
Tris*HCI, 2 mM MgCI2, and adjusting the pH to 8.5 reduces
the recognition specificity of the EcoRI endonuclease to the
tetranucleotide sequence, (NAATTN)The enzymatic
d(N-T-T-A-A-N)' h nyai
activity responsible for this substrate recognition isreferred
to as EcoRI*. Cleavage of pVH51 plasmid DNA under
EcoRI* conditions results in a number of partial digest fragments, some of which disappear slowly over a prolonged digestion period. This suggests that different recognition sites
are cleaved at different rates. Comparison of DNA fragment
patterns of modified and unmodified pVH51 DNA indicates
that the canonical EcoRI sequence is the mostrapidly
cleaved site under EcoRI* conditions. DNA modified in vivo
by the EcoRI methylase is not cleaved by the EcoRI endonuclease under standard conditions, but is cleaved under
EcoRI* conditions at sites other than the standard EcoRI
substrate.
Type II restriction endonucleases and modification methylases are widespread in the microbial world. The substrate
specificities of a number ofthese enzymes have been determined (1-4). The substrates are symmetrical DNA sequences of 4 to 6 nucleotide base pairs. The EcoRI restriction endonuclease and modification methylase recognize
and enzymatically alter the symmetrical sequence

the substrate recognition of the EcoRI endonuclease to the

tetranucleotide, d(N-TAT-A-A-N) In this paper we present a
preliminary analysis of thisactivity, 'referred to as the
EcoRI* activity of the EcoRI endonuclease.

d(N-G-A-A-T-T-C-N) (5, 6) where the arrows designate the
d(N-C-T-T-A-A-G-N)
positions of phosphodiester bond cleavage and the asterisks
designate methylated nucleotides. These reactions occur at
the level of duplex DNA, without involvement of cruciform
or "hairpin" structural rearrangements of the polynucleotide strands(Greene et al., manuscript submitted).
Under conditions producing the maximum rate of endonucleolytic cleavage of unmodified DNA the EcoRI endonuclease yields limit digests (7-9). Specific alteration of the
standard EcoRI endonuclease reaction conditions reduces
Abbreviations: dN, any of the four standard deoxyribonucleosides;
NP40 is Nonidet P40, a nonionic detergent from Shell ChemicalCompany; SV40, simian virus 40; col El, colicin El plasmid; rRi
and mRI, RI host restriction and modification phenotypes; ASV,
avian sarcoma virus.

3310

MATERIALS AND METHODS
Strains and Plasmids. Escherichia coli strain HB129 was
derived from an endonuclease I deficient E. coli 1100 (10,
11). MB100 was derived from HB129 by transformation
with the plasmid pMBl (molecular weight 5.5 X 106daltons), which is similar to colicin El (col El) (12) except that
it carries an additional 1.3 X 106 dalton piece of DNA containing the EcoRI restriction and modification genes (M. C.
Betlach, unpublished observation). Strain MB101 was derived from HB129 by introduction of the pMB2 plasmid,
which was derived from pMB1 by HindIII endonuclease digestion of pMB1 and removal of a fragment...
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