Restriction Enzymes 113there is no sequence similarity over an extended stretch of amino
acids. Lauster (52), however, has detected intra- and intermolecular
homologies comprising 10-20 amino acid residues: BsuRI, PaeR7,
and PstI each contain a twofold repeat. BsuRI in addition contains a
fourfold repeat that is homologous to regions found also in EcoRI,
EcoRV, and PaeR7. Most restriction enzymes are active as dimers of
identical subunits with subunit mol mass ranging from approx 25,000
to 35,000 Da, exceptions being BsuBI, BsuFI, BsuRI, EcoRII, and
FokI with mol mass of 43,000, 46,000, 66,000, 45,000, and 67,000 Da,
respectively. The BsuRI enzyme, which has been reported to be active
as a monomer, might be--as suggested by sequence repetitions--a
quasi dimer. The FokI enzyme differs from most type II restriction
enzymes, in as much as it recognizes an asymmetric sequence. A few
restriction enzymes have been reported to be small and monomeric,
e.g., BglI, BspRI, and Pall (53-55). These differences in quarternary
structure may reflect major differences in reaction mechanisms, or
that the state of aggregation depends critically on the absence or pres-
ence of the substrate.
The three-dimensional structure of two restriction enzymes, EcoRI
and EcoRV, has been solved (29,56; Winkler unpublished). Work on
crystallographic analysis of HhalI is in progress (57). Although there
is no similarity in the topological arrangement of the secondary struc-
ture elements of EcoRI and EcoRV, the overall shape is similar. In both
cases, the two identical subunits are arranged such that a deep cleft is
formed that--as is evident from the X-ray structure analysis of the
protein DNA cocrystals--constitutes the DNA binding site. It is note-
worthy that both EcoRI and EcoRV form complexes with twofold
symmetry, which means that the two identical subunits of these restriction
enzymes are engaged in the same set of interactions with the two
halves of their palindromic recognition sequences, as originally sug-
gested by Kelly and Smith (58).
2.3. Reaction Mechanisms
The presence of Mg 2+ ion is essential for the enzymatic activity of
all restriction enzymes. For EcoRI, it was shown that other divalent
cations can be used: Mn 2÷, Co 2÷, and Zn 2÷ (59). Presumably, Mg 2÷ parti-
cipates in the catalytic event by polarizing the phosphodiester bond to
be cleaved and/or activating water to form the required nucleophile.