Report 2008-2010

Structure and function of restriction endonucleases: projects
overview
DNA synapsis through transient tetramerization triggers cleavage
by Ecl18kI restriction enzyme.
To cut DNA at their target sites, restriction enzymes assemble into different
oligomeric structures. The Ecl18kI endonuclease in the crystal is
arranged as a tetramer made of two dimers each bound to a DNA
copy. However, free in solution Ecl18kI is a dimer. To find out whether
the Ecl18kI dimer or tetramer represents the functionally important assembly,
we generated mutants aimed at disrupting the putative dimerdimer
interface and analysed the functional properties of Ecl18kI and
mutant variants. We show by atomic force microscopy that on twosite
DNA, Ecl18kI loops out an intervening DNA fragment and forms
a tetramer. Using the tethered particle motion technique, we demonstrate
that in solution DNA looping is highly dynamic and involves a
transient interaction between the two DNA-bound dimers. Furthermore,
we show that Ecl18kI cleaves DNA in the synaptic complex
much faster than when acting on a single recognition site. Contrary to
Ecl18kI, the tetramerization interface mutant R174A binds DNA as a
dimer, shows no DNA looping and is virtually inactive. We conclude
that Ecl18kI follows the association model for the synaptic complex assembly
in which it binds to the target site as a dimer and then associates
into a transient tetrameric form to accomplish the cleavage
reaction.
Figure 2. Reaction pathway of the Ecl18kI restriction
enzyme on the two-site DNA. At
Ecl18kI concentrations much below that of the
DNA, a single Ecl18kI dimer presumably binds
to only one individual target site. Binding of
the second dimer at increased enzyme concentrations
produces an unlooped protein-
DNA complex, where two dimers act on the
two DNA sites independently. Tetramerization
of two DNA-bound Ecl18kI dimers results in
the looped synaptic complex, which is optimal
for catalysis and gives in fast cleavage.
A novel mechanism for the scission of double-stranded DNA: BfiI
cuts both 3'-5' and 5'-3' strands by rotating a single active site.
Metal-dependent nucleases that generate double-strand breaks in
DNA often possess two symmetrically-equivalent subunits, arranged
so that the active sites from each subunit act on opposite DNA
strands. Restriction endonuclease BfiI belongs to the phospholipase
D (PLD) superfamily and does not require metal ions for DNA cleavage.
It exists as a dimer but has at its subunit interface a single active
site that acts sequentially on both DNA strands. The active site contains
two identical histidines related by 2-fold symmetry, one from
each subunit. This symmetrical arrangement raises two questions: first,
what is the role and the contribution to catalysis of each His residue;
secondly, how does a nuclease with a single active site cut two DNA
strands of opposite polarities to generate a double-strand break. In
this study, the roles of active-site histidines in catalysis were dissected
by analysing heterodimeric variants of BfiI lacking the histidine in one
subunit. These variants revealed a novel mechanism for the scission
of double-stranded DNA, one that requires a single active site to not
only switch between strands but also to switch its orientation on the
DNA.
Figure 3. A model for the reactions of WT BfiI on the bottom and the top
strand of a DNA duplex. The H105 residue from the same subunit of the
homodimer, the 2° subunit not bound to the DNA makes the nucleophilic
attacks on the target phosphodiester bonds in both bottom and top strands
of the DNA. To match the anti-parallel orientation of the two strands, the
N-terminal domains of BfiI must rotate by 180° between the two hydrolysis
reactions.
35 th anniversary
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