Chemistry Reference
In-Depth Information
previous IOP conjugate, which allows the OP-Cu complex to extend and cleave the
adjacent bases of the DNA substrate. These artificial cleavage enzymes could ulti-
mately be used to identify the number, position and orientation of NarL monomers
on several promoters and may be extended to other regulatory proteins. These results
highlight the promise of chemical nucleases as tools for biological investigation.
5.2.4
Additional Applications for OP Conjugates
Other research groups have also created artificial nucleases by OP conjugation. John-
son and co-workers created an artificial nuclease, which aided in the understanding of
DNA conformational changes upon protein binding [32]. E. coli Fis (factor for inver-
sion stimulation) was chosen as the protein scaffold and binds to DNA with a low
sequence specificity. X-Ray crystallography and electrophoretic mobility studies indi-
cated significant bending of the DNA upon Fis binding [33, 34]. To aid in the study of
DNA-Fis complexes, OP-Cu was conjugated to four separate mutated cysteines. Of
these four conjugates, N98C-OP and N78C-OP displayed nuclease activity. Scission
patterns from these two nucleases not only confirmed earlier studies of DNA bending,
but also revealed that the DNA wrapped around the Fis dimer.
The E. coli catabolite gene activator protein (CAP) binds to a 22 base-pair, two-fold
symmetric DNA recognition site with high affinity (K
A
=4
10
10
M
-1
) [35]. Ebright
and co-workers successfully converted CAP into a site-specific DNA cleavage enzyme.
In their first attempt, OP was conjugated to the only solvent accessible cysteine (C
178
)
of CAP. The catalytic group was placed at this position so that intra-CAP interactions
would not be disrupted, CAP-DNA contacts would not be comprised, and OP would be
in close proximity for a favorable attack on the phosphodiester bond. The binding con-
stant for this conjugate was 1
10
8
M
-1
, indicating that the DNA binding ability of CAP
was not significantly lost due to conjugation with OP. The CAP178-OP conjugate
cleaved the DNA site at four adjacent nucleotides (Figure 5.4).
Earlier work indicated that in the specific CAP-DNA complex the DNA was bent 90
8
away from the protein but was not distorted in the nonspecific complex. (CAP bound
to its noncognate DNA site) [3, 36]. This DNA bending phenomenon was exploited to
construct a new CAP-OP nuclease capable of single site cleavage [37]. The crystal struc-
ture of the specific CAP-DNA complex revealed that amino acids 24-26 and 89-91 of
CAP were close to the DNA substrate [38]. With this in mind, residue 26 was mutated
Figure 5.4 DNA cleavage sites produced by the
CAP178-1,10-phenanthroline conjugate. Longer ar-
rows indicate sites where greater cleavage efficiency
occurred.
