E.
coli DNA Polymerase III
Beta Subunit:
The Sliding DNA Clamp
David Marcey
© 2006
I.
Introduction
II. Monomer and Dimer Structure
III. Interactions with DNA
IV. References
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I.
Introduction
The
remarkable, donut-shaped molecule to your left is the beta subunit
of DNA polymerase III of E. coli (pol III). This subunit provides
for the remarkable processivity of the holoenzyme during DNA replication.
Processivity refers to ability of polymerases to add many hundreds
or thousands of nucleotides to a growing chain without dissociating
from the template. Processivity partially accounts for the rapid rates
of DNA synthesis by DNA polymerases. For example, E. coli replicates
its entire genome in ~40 minutes (~80,000 bp/min). The pol III beta
subunit is a ring-shaped clamp that embraces DNA in a central 35 angstrom
hole, tethering the remainder of pol III to the template.
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II. Monomer and
Dimer Structure
The
beta subunit is a homodimer of two, 366 amino acid monomers,
each monomer providing one half of the clamp.
The
dimer interface is a novel continuation, across the monomer boundary, of a beta sheet
structure, indistinguishable from intra-monomer beta sheets (one interface
is illustrated here). In addition to four strong hydrogen bonds that
link the beta strands across the interface, there are several other
linkages helping to stabilize the dimer, including:
hydrophobic
interactions of amino acid sidechains - R
groups of phe106
and ile108
of one monomer pack against ile272
and leu273
of the other and form a hydrophobic core;
ionic bonds (salt bridges) between four pairs
of amino acid side chains exposed to solvent (water);
ionic bond pairs (arg96-glu300
and arg103-glu304)
that are inaccessible to solvent and that likely form
particularly strong ionic bonds.
The
beta subunit monomers are arranged in a head-to-tail (N-C-->N-C-->)
orientation. This produces non-symmetric faces of the dimeric ring.
The
two carboxy
termini project from the face that binds
the remainder of the Pol III holoenzyme. Note that this face contains
prominent loops that are well-suited
to bind other pol III subunits.
Each
monomer comprises three domains with nearly identical structure, but
not identical amino acid sequence. The
amino,
central,
and carboxy
domains each harbor an outer layer of two beta
sheets that support 2 inner alpha helices.
Thus,
the core of the dimeric clamp is lined with 12
alpha helices (2 helices/domain x 3 domains/monomer
x 2 monomers).
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III.
Interaction with DNA
The 35 Angstrom hole of the beta dimer is large
enough to accommodate double helical nucleic acid with little steric
hindrance as modeled here for B-DNA (~20 Angstrom diameter). The tilt
of the
12 central alpha helices is
similar due to the symmetrical arrangement of the six domains. The
axis of each alpha helix can be seen to be perpendicular to the sugar-phosphate
backbone of both major and minor DNA grooves when the DNA is
modeled perpendicular to the plane of the beta clamp ring.
Many DNA-binding proteins contain alpha helices that are oriented
parallel to the nucleic acid backbone. This orientation allows the
alpha helices to recognize and fit into the major groove of target
DNA. In contrast, the perpendicular orientation of the beta clamp
helices and DNA
backbone seems designed to prevent access of the protein to
either DNA groove and therefore to facilitate rapid sliding of the
clamp along the DNA axis.
These
principles hold for interaction with A-form DNA-RNA duplexes (~25
Angstrom diameter), found at the site of initial clamping of the Beta
subunit at the RNA-primed template of the start of an Okazaki fragment.
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IV.
References
Kong,
X-P., Onrust, R., O'Donnell, M., and J. Kuriyan (1992). Three-Dimensional
Structure of the Beta Subunit of E. coli DNA Polymerase III
Holoenzyme: A Sliding DNA Clamp. Cell 69: 425-437.
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