SEQUENCE-SPECIFIC DNA-BINDING BY ECOKI, A TYPE IA DNA RESTRICTION ENZYME

The type I DNA restriction and modification enzymes of prokaryotes are multimeric enzymes that cleave unmethylated, foreign DNA in a complex process involving recognition of the methylation status of a DNA targ et sequence, extensive translocation of DNA in both directions towards the enzyme bound at the target sequence, Am hydrolysis, which is beli eved to drive the translocation possibly via a helicase mechanism, and eventual endonucleolytic cleavage of the DNA. We have examined the DN A binding affinity and exonuclease III footprint of the EcoKI type LA restriction enzyme on oligonucleotide duplexes that either contain or lack the target sequence. The influence of the cofactors, S-adenosyl m ethionine and Am, on binding to DNA of different methylation states ha s been assessed. EcoKI in the absence of Am, with or without S-adenosy l methionine, binds tightly even to DNA lacking the target site and th e exonuclease footprint is large, approximately 45 base-pairs. The pro tection is weaker on DNA lacking the target site. Partially assembled EcoKI lacking one or both of the subunits essential for DNA cleavage, is unable to bind tightly to DNA lacking the target site but can bind tightly to the recognition site. The addition of ATP to EcoKI, in the presence of AdoMet, allows tight binding only to the target site and t he footprint shrinks to 30 basepairs, almost identical to that of the modification enzyme which makes up the core of EcoKI. The same effect occurs when S-adenosyl homocysteine or sinefungin are substituted for S-adenosyl methionine, and ADP or ATP IS are substituted for ATP. It i s proposed that the DNA binding surface of EcoKI comprises three regio ns: a ''core'' region which recognises the target sequence and which i s present on the modification enzyme, and a region on each DNA cleavag e subunit. The cleavage subunits make tight contacts to any DNA molecu le in the absence of cofactors, but this contact is weakened in the pr esence of cofactors to allow the protein conformational changes requir ed for DNA translocation when a target site is recognised by the core modification enzyme. This weakening of the interaction between the DNA cleavage subunits and the DNA could allow more access of exonuclease III to the DNA and account for the shorter footprint. (C) 1998 Academi c Press.