Sliding clamps have been discovered
in all types of organisms. In E.
coli, the sliding clamp is referred to as the ß subunit,
in T4 bacteriophage it is referred to as gp43, and in eukaryotes the
sliding clamp is called PCNA, which stands for Proliferating Cell
|ß clamp, homodimer
|gp43, homotrimer of gene 43 protein
PCNA of eukaryotes requires three
monomers to form a typical dough-nut like structure. The
homotrimer is composed of six repeating domains in six-fold
symmetry. PCNA has a two layer structure composed of 12 inside
alpha-helices and outside beta-sheets. Crystallography reveals
that the circular sliding clamp has an opening of 30-35 Å, large
enough to accomodate the double-stranded DNA.
The inside channel of the ring is positively charged
and lined with alpha-helices that do not make any specific contact with
DNA, thus allowing the sliding clamp to slide freely on the duplex
behind the polymerase. The half-life of PCNA-DNA complex is 23
minutes and PCNA can be reused for multiple rounds of DNA
Schematic representation of
SLIDING CLAMP LOADER:
The task of loading and unloading of
sliding clamps on the DNA strand is being performed by the clamp
loaders. The clamp loaders in E.
coli, T4 bacteriophage, and eukaryotes are all composed of five
subunits. In eukaryotes, the clamp loader is called Replication
Factor C (RF-C). The five subunits of RF-C are called RFC-A,
RFC-B, RFC-C, RFC-D, and RFC-E, belonging to AAA+ family of
|gamma complex consisting of 5 subunits
|gene 44/62 protein complex consisting of 5 subunits
|RF-C consisting of 5 subunits A, B, C, D and E
The RF-C complex is seated on top of a closed PCNA
ring, but slightly tipped away from it. Each RF-C is further
divided into three domains; Domain I, Domain II, and Domain III.
Domain I is a RecA-type ATPase domain; while Domain II is a helical
domain that is characteristic of AAA+ ATPases. Together the
Domain I and II form the AAA+ module, which in turn is connected by a
flexible linker to the helical Domain III. The Domain III of all
five subunits of RF-C form a cylindrical structure which is called a
"collar." The five AAA+ modules form a right hand spiral
resulting in connection of only three subunits (RFC-A, RFC-B, and
RFC-C) with the PCNA. This leaves a wedge-shaped gap between
RFC-E and PCNA. The spiral assembly of RFC-A, RFC-B, RFC-C, and
RFC-D is being held together by ATP gamma-S, nucleotides that anchor
intersubunit interactions through hydrogen bonds to the phosphate
groups. Domain IV of RFC-A, which is located between RFC-A and E,
provides a physical link between the two ends of the RF-C spiral.
Five subunits of RFC
PCNA interacts with RFC with its
hydrophobic grooves, two of which are engaged by RFC-A and RFC-C.
High sequence similarity between the clamp loaders suggest that their
mechanism of action may be very similar.
DNA polymerases, the enzymatic bodies that catalyze the
addition of a nucleotide onto an existing 3'-OH of a growing DNA chain,
are rather poor at staying on task. They synthesize short pieces
of DNA, but tend to fall off the template DNA before getting too
far. This definitely does not help when an entire genome needs to
be copied. DNA polymerases require tethering to an accessory
factor, a ring-shaped clamp, to remain bound to DNA during
replication. Since the clamp cannot operate on its own, the help
of the clamp loader opens up the clamp so that it can become wrapped
around the DNA, and also unloads the clamp so that the polymerase can
dissociate at the appropriate time.
Above you can get a clear visualization of replication. As you
can see, the sliding clamp is holding each polymerase unit to its
template by physically encircling the template DNA strand.
DNA CLAMP-CLAMP LOADER
MECHANISM OVERVIEW IN E. COLI:
Since the DNA clamp-clamp loader
process is difficult to understand, the use of the E. coli modef for
the function of the clamp loader and clamp, helps to get a better
understanding when it comes to visualizing the mechanism in higher
organisms, such as eukaryotes.
*HOW DOES THE CIRCULAR CLAMP MOLECULE WRAP ITSELF
AROUND DNA TO BEGIN THE PROCESSIVE SYNTHESIS?
For E. coli, the clamp
loader is known as the gamma-complex, which was noted for being made up
of 5 subunits. The beta-subunit is known as the actual clamp
itself and converts DNA polymerase III from a distributive enzyme to a
highly processive one, which remains bound through thousands of
incorporation reactions. The gamma-complex (clamp loader)
actually binds to the beta-subunit (clamp) and carries out the ring
opening that leads to clamp attachment. ATP is required, but ATP
does not have to undergo hydrolysis for opening the clamp or
positioning around the DNA. A conformational change driven by ATP
binding leads the complex to bind DNA and the gamma-complex hydrolyzes,
which then allows the clamp to close. This happens once per round
of replication on the leading strand, but on the lagging strand, the
polymerase must rebind at the initation of synthesis of each Okazaki
fragment. The clamp must dissociate when the 5' end of the
pre-existing daughter DNA strand is reached. Clamp loading must
occur continuously and rapidly, with the clamp loader also unloading
coli clamp loading complex mechanism
EUKARYOTIC DNA CLAMP
The head to tail connection at the
interface of clamp monomers is maintained by stable interactions of
hydrophobic residues. The clamp loaders function is to temporally
open the sliding clamp to allow DNA strands to pass through. In
this case, we will be talking about RFC as the "clamp loader."
RFC forms a complex with PCNA and the interaction mode is changed by
ATP. To transfer DNA into the center of the PCNA clamp, there is
a common ATP dependent mechanism involved. Without having ATP,
the RFC loader is relatively weak. The binding of ATP to RFC
allows the PCNA ring to open. If its hydrolysis occurs, a stable
clamp is formed on the DNA and the RFC-PCNA complex is disrupted.
RFC always loads the PCNA on DNA to face its C-side to DNA synthesis
direction. They are oriented appropriately with respect to the 3'
end of the primer strand.
Loading and unloading pathway of PCNA at
the 3' end of DNA by interaction with RFC.
delta is the principle leading strand polymerase. The RFC-PCNA
complex is essential and sufficient for the loading and polymerase
delta on DNA and for the translocation of the enzyme along the
gap. Polymerase delta requires PCNA to help it carry out highly
processive DNA synthesis. PCNA plays a comparable role to that of
the beta-subunit of E.coli
DNA polymerase III. After completion of its task as a polymerase
delta processivity factor, PCNA needs to be released from DNA.
RFC causes the unloading of PCNA through the hydrolysis of ATP as the
reverse reaction of loading.
SLIDING CLAMP FUNCTIONS:
PCNA is required for the elongation
stage of DNA replication especially for synthesis of the leading and
lagging strands at a continuous rate. Without the help of PCNA,
unligated short Okazaki-DNA fragments would accumulate due to the
requirement of PCNA and polymerase delta for complete synthesis.
PCNA is the processivity factor for polymerase delta. Without
PCNA, the polymerase alone would only be able to elongate a short
number of nucleotides from primers.
PCNA has been recognized to also be helpful in DNA
repair, which involves DNA re-syntheses which occurs after removal of
DNA lesions. A need for PCNA has been reported for nucleotide
excision repair, base excision repair, and mismatch repair. In
general, PCNA is a DNA repair factor involved in DNA damage recognition
and DNA re-synthesis steps through interactions with multiple repair
factors. There is also evidence between PCNA and cell cycle
The expression of the RFC-PCNA complex could
definitely become an enormous source of information, leading to a
better understanding of this fascinating protein. Due to these
linked contributions of PCNA in other cellular functions, PCNA will
eventually become a "master molecule" for chromosome processing, rather
than just a "clamp."
Crystal structure of the Eukaryotic Clamp Loader (RFC) bound to the DNA
Sliding Clamp (PCNA)
Bowman, Gregory D., et al. 2004. Structural analysis of a
eukaryotic sliding DNA clamp-clamp loader complex. Nature. Vol. 429, p.724-730.
Ellison, Viola and Bruce Stillman. 2001. Opening of the
Clamp: An Intimate View of an ATP-Driven Biological Machine. Cell. Vol. 106, p.655-660.
Jeruzalmi, David, et al. 2002. Clamp loaders and sliding
clamps. Current Opinion in
Structural Biology. Vol. 12, p. 217-224.
Mathews, Christopher K., et al. Biochemistry. 3rd ed.
Addison Wesley Longman, Inc. San Francisco, CA. 2002.
Mossi, Romina and Ulrich Hubscher. 1998. Clamping down on
clamps and clamp loaders: The eukaryotic replication factor C. European Journal of Biochemistry.
Vol. 254, p. 209-216.
Tsurimoto, Toshiki. 1999. PCNA Binding Proteins. Frontiers in Bioscience. Vol.
4, p. 849-858.