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 DNA element with affinity for the activator site of the enzyme: a cleavage-enhancer DNA element. Measurements of the mobility of NaeI activity in comparison with protein standards on gel permeation columns and glycerol 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 cleavage, depending upon NaeI concentration, and reduced the NaeI concentration required for the transition from nicking to cleavage activity. A gel mobility-shift assay of the interaction of NaeI with enhancer showed the formation of two complexes. Results using different sized DNAs and different percentage acrylamide gels for gel mobility-shift analysis implied that the two complexes were caused by NaeI monomer and dimer structures rather than one and two DNA binding. Dimer formation increased with the affinity of enhancer for NaeI. UV cross-linking "captured" the NaeI-enhancer complex; electrophoretic analysis of the cross-linked products showed NaeI dimer bound to enhancer. These results imply a model for cleavage enhancement in which enhancer binding stabilizes an active NaeI dimer conformation ("cleavasome") that cleaves both DNA strands before dissociating.
Baxter
B K
BK
Department of Pathology and Curriculum in Genetics, University of North Carolina, Chapel Hill 27599-7295.
Topal
M D
MD
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D012091:Repetitive Sequences, Nucleic Acid; D014466:Ultraviolet Rays
D046911:Macromolecular Substances; D004247:DNA; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific