closed thermodynamic cycle, the particular pathway chosen does not affect the equilibrium affinity. However,
the second (monomer) pathway has been noted to have faster association rates for certain leucine-zipper
proteins.^26
Some proteins find their DNA target sites at rates approaching 10^3 -fold greater than those expected if
site location were diffusion-controlled.^27 Three experimentally determined mechanisms account in part for
this apparent acceleration (Figure 10.12).
1.sliding– involves diffusion in one dimension,
2.hopping/jumping– involves limited sliding diffusion, followed by dissociation and re-association
at a different point on the DNA chain,^28 and
3.inter-segmental transfer– allows the direct transfer from one segment of DNA to another so as to
slide or hop to different regions.^29
In all cases, the rate enhancement is expected to be a factor of only sixfold over diffusion-controlled rates,
since the diffusion rates are proportional to twice the dimensions of the search space. Most of the apparent
rate acceleration may be due to electrostatic attraction, because of the polyelectrolyte character of the DNA.
In the cell, DNA is hardly ever in a free form. Thus, sliding-type diffusion is extremely limited, for example
because of obstacles such as nucleosomes (Section 10.6.1). However, three-dimensional diffusion mech-
anisms such as hopping provide a means to maintain the search process even with such obstacles present.^30
10.5 The Specificity of DNA Enzymes
10.5.1 Restriction Enzymes: Recognition through the Transition State
Restriction enzymes are bacterial proteins that provide defence against foreign DNA. They recognize and
cleave specific DNA sequences on both strands. Many, but not all, have twofold symmetry and so recog-
nize palindromic sites. The enzymes recognize typically a sequence of four to eight base pairs and, not sur-
prisingly, have extremely high specificity for well-defined target sites on DNA. Any relaxed specificity
would have dire consequences for a bacterium, since its own DNA would be at risk of cleavage at sites not
protected by its own methylase enzyme (Section 5.3.1).
The mechanism of restriction enzyme cleavage of DNA involves nucleophilic attack of a hydroxide ion
on the scissile phosphorus atom to generate a 5-phosphate group (e.g.for the enzyme HincII). The hydroxyl
ion attack causes an in-line displacement of the 3-hydroxyl group viaa penta-coordinate transition state.
Restriction enzymes require divalent cations for activity, which are used to activate the water molecule for
attack and to stabilize the negative charge that develops transiently on the transition state.^31
It might seem paradoxical that many restriction enzymes bind target and non-target DNA with similar
affinities. Therefore, specificity must arise at the catalytic step, probably because of the distortions
induced in the transition state. Such distortions are seen in the crystal structure of the EcoRI restriction
404 Chapter 10
Figure 10.12 Possible modes for search mechanisms of DNA-binding proteins for their target sites