Binding and recognition of GATATC target sequences by the EcoRV restriction endonuclease: a study using fluorescent oligonucleotides and fluorescence polarization.

Abstract:

:Oligonucleotides labeled with hexachlorofluorescein (hex) have enabled the interaction of the restriction endonuclease EcoRV with DNA to be evaluated using fluorescence anisotropy. The sensitivity of hex allowed measurements at oligonucleotide concentrations as low as 1 nM, enabling K(D) values in the low nanomolar range to be measured. Both direct titration, i.e., addition of increasing amounts of the endonuclease to hex-labeled oligonucleotides, and displacement titration, i.e., addition of unlabeled oligonucleotide to preformed hex-oligonucleotide/EcoRV endonuclease complexes, have been used for K(D) determination. Displacement titration is the method of choice; artifacts due to any direct interaction of the enzyme with the dye are eliminated, and higher fluorescent-labeled oligonucleotide concentrations may be used, improving signal-to-noise ratio. Using this approach (with three different oligonucleotides) we found that the EcoRV restriction endonuclease showed a preference of between 1.5 and 6.5 for its GATATC target sequence at pH 7.5 and 100 mM NaCl, when the divalent cation Ca2+ is absent. As expected, both the presence of Ca2+ and a decrease in pH value stimulated the binding of specific sequences but had much less effect on nonspecific ones.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Reid SL,Parry D,Liu HH,Connolly BA

doi

10.1021/bi001956p

subject

Has Abstract

pub_date

2001-02-27 00:00:00

pages

2484-94

issue

8

eissn

0006-2960

issn

1520-4995

pii

bi001956p

journal_volume

40

pub_type

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