The specific binding, bending, and unwinding of DNA by RsrI endonuclease, an isoschizomer of EcoRI endonuclease.
To determine whether RsrI endonuclease recognizes and cleaves the sequence GAATTC in duplex DNA similarly to its isoschizomer EcoRI we initiated a functional comparison of the two enzymes. Equilibrium binding experiments showed that at 20 degrees C RsrI endonuclease binds to specific and nonspecific sequences in DNA with affinities similar to those of EcoRI. At 0 degrees C the affinity of RsrI for its specific recognition sequence is reduced 7-fold whereas the affinity for noncanonical sequences remains relatively unchanged. Unlike EcoRI, incubation of RsrI endonuclease with N-ethylmaleimide inactivates the enzyme; however, preincubation with DNA prevents the inactivation. The N-ethylmaleimide-treated enzyme fails to bind DNA as assayed by gel mobility shift assays. Comparison of the deduced amino acid sequences of RsrI and EcoRI endonucleases suggests that modification of Cys245 is responsible for the inactivation. Fe(II). EDTA and methidiumpropyl-EDTA.Fe(II) footprinting results indicate that RsrI, like EcoRI, protects 12 base pairs from cleavage when bound to its specific recognition sequence in the absence of Mg2+. RsrI bends DNA by approximately 50 degrees, as determined by measuring the relative electrophoretic mobilities of specific RsrI-DNA complexes with the binding site in the center or near the end of the DNA fragment. This value is similar to that reported for EcoRI. RsrI also unwinds the DNA helix by 25 degrees +/- 5 degrees, a value close to that reported for EcoRI endonuclease. Collectively, these results indicate that the overall structural changes induced in the DNA by the binding of RsrI and EcoRI endonucleases to DNA in the absence of Mg2+ are similar. In the accompanying paper (Aiken, C. R., McLaughlin, L. W., and Gumport, R. I. (1991) J. Biol. Chem. 266, 19070-19078) we present results of studies of RsrI endonuclease using oligonucleotide substrates containing base analogues which suggest differences in the ways the two enzymes cleave DNA.
Aiken
C R
CR
Department of Biochemistry, College of Medicine and School of Chemical Sciences, University of Illinois, Urbana 61801.
Fisher
E W
EW
Gumport
R I
RI
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004247:DNA; D015246:Deoxyribonuclease EcoRI; D005033:Ethylmaleimide