Copyright © 2004 Cell Press. All rights reserved.
Molecular Cell, Vol 5, 889-895, May 2000
Structure of BamHI Bound to Nonspecific DNA: A Model for
Hector Viadiu 12 and Aneel
K. Aggarwal 2¦
1. Department of Biochemistry
and, Molecular Biophysics, Columbia University, New York, New
York 10032, USA
2. Structural Biology Program, Department of Physiology and Biophysics,
Mount Sinai School of Medicine, New York, New York 10029, USA
The central problem faced by DNA binding proteins is how to select
the correct DNA sequence from the sea of nonspecific sequences
in a cell. The problem is particularly acute for bacterial restriction
enzymes because cleavage at an incorrect DNA site could be lethal.
To understand the basis of this selectivity, we report here the
crystal structure of endonuclease BamHI bound to noncognate
DNA. We show that, despite only a single base pair change in
the recognition sequence, the enzyme adopts an open configuration
that is on the pathway between free and specifically bound forms
of the enzyme. Surprisingly, the DNA drops out of the binding
cleft with a total loss of base-specific and backbone contacts.
Taken together, the structure provides a remarkable snapshot
of an enzyme poised for linear diffusion (rather than cleavage)
along the DNA.
We report here the structure of BamH I bound to a noncognate
DNA sequence (GAATCC) that differs by only a single base pair
from the cognate (G G ATCC) sequence. The structure reveals the
enzyme in a distinct conformation that is incompetent for cleavage
but competent for sliding.
BamHI Forms a Loose, Dynamic Complex with Nonspecific DNA
The noncognate DNA is accommodated
loosely within a cleft at the bottom of the BamH I dimer
. It protrudes out of the cleft, whereas, in the specific complex,
the DNA is almost surrounded by the enzyme. The two-fold symmetry
of BamH I coincides with the approximate two-fold axis
of the DNA. However, compared to the specific complex, the enzyme
is tilted about this axis by about 20°, resulting in markedly
different DNA binding surfaces in the two complexes.
Moreover, in the nonspecific complex, the DNA binding cleft is
widened by an outward symmetrical motion of the BamHI monomers
(R and L), such that the distance across the cleft increases
from 20 Å to 25 Å. Correspondingly, the buried solvent-accessible
surface area decreases dramatically from 4350 Å2 to 1489
Å2 in going from the specific to the nonspecific structure.
The DNA remains primarily B form (as in the specific complex),
though it is substantially frayed at the ends. Remarkably, all
of the base-specific interactions and DNA backbone contacts are
I Bound to Nonspecific DNA
The structure viewed down the DNA axis. The alpha helices are
colored in green, the beta strands in purple, and the DNA in
orange. Only one subunit is labeled. N and C mark the N and C
termini of the protein.
Structures of free (A), nonspecific
(B), and specific (C) DNA-bound forms of BamH I.
Regions that undergo local conformational
changes are shown in yellow color. The enzyme becomes progressively
more closed around the DNA as it goes from the nonspecific to
the specific DNA binding mode. Residues 7992 are unstructured
in the free enzyme but become ordered in both the nonspecific
and specific DNA complexes, albeit in different conformations.
The C-terminal residues unwind in the specific complex to form
partially disordered arms, whereas in the nonspecific complex
they remain alpha helical