FORMATION OF A CLEAVASOME - ENHANCER DNA-2 STABILIZES AN ACTIVE CONFORMATION OF NAEI DIMER

Cleavage of DNA by NaeI-type restriction enzymes is stimulated by a DN A element with affinity for the activator site of the enzyme: a cleava ge-enhancer DNA element. Measurements of the mobility of NaeI activity in comparison with protein standards on gel permeation columns and gl ycerol gradients demonstrated that NaeI, without enhancer, can form a 70 000 MW dimer. The dimer, however, is inactive: it could not cleave the ''resistant'' NaeI site in M13mp18 DNA in the absence of enhancer. In cleavage assays, enhancer stimulated either DNA nicking or DNA cle avage, depending upon NaeI concentration, and reduced the NaeI concent ration required for the transition from nicking to cleavage activity. A gel mobility-shift assay of the interaction of NaeI with enhancer sh owed the formation of two complexes. Results using different sized DNA s and different percentage acrylamide gels for gel mobility-shift anal ysis implied that the two complexes were caused by NaeI monomer and di mer structures rather than one and two DNA binding. Dimer formation in creased with the affinity of enhancer for NaeI. UV cross-linking ''cap tured'' the NaeI-enhancer complex; electrophoretic analysis of the cro ss-linked products showed NaeI dimer bound to enhancer. These results imply a model for cleavage enhancement in which enhancer binding stabi lizes an active NaeI dimer conformation (''cleavasome'') that cleaves both DNA strands before dissociating.