Jd. Colandene et Md. Topal, THE DOMAIN ORGANIZATION OF NAEI ENDONUCLEASE - SEPARATION OF BINDING AND CATALYSIS, Proceedings of the National Academy of Sciences of the United Statesof America, 95(7), 1998, pp. 3531-3536
NaeI is a remarkable type II restriction en donuclease. It must bind t
wo recognition sequences to cleave DNA, forms a covalent protein-DNA i
ntermediate, and is only 1 aa change away from topoisomerase and recom
binase activity, The latter activities apparently derive from reactiva
tion of a cryptic DNA ligase active site, Here, we demonstrate that Na
eI has two protease-resistant domains, involving approximately the N-t
erminal and C-terminal halves of the protein, linked by a protease-acc
essible region of 30 aa, The domains were purified by cloning, The C-t
erminal domain was shown by gel mobility-shift assay to have approxima
tely 8-fold lower DNA-binding ability than intact NaeI, Analytical ult
racentrifugation showed this domain to be a monomer in solution, The N
-terminal domain, which contains the catalytic region defined by rando
m mutagenesis, did not bind DNA and was a mixture of different-sized c
omplexes in solution implying that it mediates self-association. DNA g
reatly inhibited proteolysis of the linker region, The results identif
y the DNA-binding domain, imply that DNA cleavage and recognition are
independent and separable, and lead us to speculate about a cleft-like
structure for NaeI.