Eco RII endonuclease has two identical DNA‐binding sites and cleaves one of two co‐ordinated recognition sites in one catalytic event

• Published in: FEBS letters Vol. 425; no. 1; pp. 29 - 34
• Main Authors: Petrauskene, O.V., Babkina, O.V., Tashlitsky, V.N., Kazankov, G.M., Gromova, E.S.
• Format: Journal Article
• Language: English
• Published: 20.03.1998
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)00184-7

Eco RII is a typical restriction enzyme that cleaves DNA using a two‐site mechanism. Eco RII endonuclease is unable to cleave DNA which contains a small number of Eco RII recognition sites but the enzyme activity can be stimulated in the presence of DNA with a high frequency of Eco RII sites. To investigate the mechanism of activation, the kinetics of stimulated Eco RII cleavage has been studied. A 14 bp substrate activated the cleavage of the 71 bp substrate, containing one Eco RII 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 Eco RII cuts one of two coordinated recognition sites in one catalytic event.