Bk. Baxter et Md. Topal, FORMATION OF A CLEAVASOME - ENHANCER DNA-2 STABILIZES AN ACTIVE CONFORMATION OF NAEI DIMER, Biochemistry, 32(32), 1993, pp. 8291-8298
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.