EcoRII endonuclease has two identical DNA-binding sites and cleaves one of two co-ordinated recognition sites in one catalytic event.
FEBS Lett
; 425(1): 29-34, 1998 Mar 20.
Article
em En
| MEDLINE
| ID: mdl-9541001
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.
Buscar no Google
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Desoxirribonucleases de Sítio Específico do Tipo II
/
Proteínas de Ligação a DNA
Idioma:
En
Ano de publicação:
1998
Tipo de documento:
Article