NaeI endonuclease must bind two DNA sites for cleavage to occur. NaeI
was purified to apparent homogeneity and used to determine the rate-li
miting step for DNA cleavage and to measure NaeI's specificity for its
cognate recognition site. Steady-state cleavage by NaeI in the presen
ce of effector DNA (activated) gave values of 0.045 s(-1) and 10 nM fo
r k(cat) and K-M for M13 DNA substrate, respectively, but values of 0.
4 s(-1) and 170 nM, respectively, for an M13 DNA fragment substrate. S
ingle-turnover cleavage of M13 DNA demonstrated that DNA strand scissi
on is not rate-limiting for turnover of NaeI. Transient kinetic analys
is of M13 DNA cleavage by NaeI showed an initial burst of substrate cl
eavage that was proportional to NaeI concentration, implying that prod
uct release is rate-limiting for turnover of NaeI. The NaeI effector a
nd substrate binding sites were found to prefer cognate over noncognat
e sequences by 10(3)-fold and at least 40-500-fold, respectively, k(ca
t) for noncognate recognition sequence was at least 10(6)-fold lower t
han that for cognate. The specificity of activated NaeI, as measured b
y k(cat)/K-M, for noncognate recognition sequence was 10(8)-fold lower
than that for cognate, and over 10(11)-fold lower when the decreased
affinity for noncognate sequence at the effector binding site was take
n into account. This specificity is approximately 10(4)-fold larger th
an for any other restriction enzyme measured.