EcoRII endonuclease has two identical DNA-binding sites and cleaves one of two co-ordinated recognition sites in one catalytic event

FEBS Letters
O V PetrauskeneE S Gromova

Abstract

EcoRII is a typical restriction enzyme that cleaves DNA using a two-site mechanism. EcoRII endonuclease is unable to cleave DNA which contains a small number of EcoRII recognition sites but the enzyme activity can be stimulated in the presence of DNA with a high frequency of EcoRII sites. To investigate the mechanism of activation, the kinetics of stimulated EcoRII cleavage has been studied. A 14 bp substrate activated the cleavage of the 71 bp substrate, containing one EcoRII recognition site (trans-activation) by a competitive mechanism: the activator increased substrate binding but not catalysis. The activation increased if the substrate concentration decreased and if the activator had a lower affinity for the enzyme than the substrate. The introduction of the second recognition site into the 71 bp duplex also enabled cleavage of this substrate (cis-activation). Pyrophosphate bonds were incorporated into one of two recognition sites to switch off the cleavage of the phosphodiester bonds. Analysis of cleavage products of these modified substrates showed that EcoRII cuts one of two coordinated recognition sites in one catalytic event.

References

Dec 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·M Conrad, M D Topal
Mar 8, 1988·Biochemistry·S E Halford, A J Goodall
May 11, 1988·Nucleic Acids Research·D H KrügerH O Smith
May 11, 1988·Nucleic Acids Research·N G DolinnayaZ A Shabarova
Feb 15, 1994·Biochemical and Biophysical Research Communications·O V PetrauskeneW Guschlbauer

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Citations

Mar 28, 2008·The Journal of Biological Chemistry·Shuntaro TakahashiYoshio Okahata
Oct 25, 2003·Nucleic Acids Research·Merlind MuckeMonika Reuter
Jul 10, 2003·The Journal of Biological Chemistry·Kamini Hingorani-Varma, Jurate Bitinaite
Feb 8, 2002·The Journal of Biological Chemistry·Merlind MückeMonika Reuter
Oct 19, 2001·The Journal of Biological Chemistry·Meera SoundararajanBernard A Connolly

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