SLIDING DNA CLAMP-

CLAMP LOADER
COMPLEX

INTRODUCTION
 DNA replication is a process that involves unwinding of the parental DNA strand,
incorporation of nucleotide precursors and renaturation of replicated molecules.
These processes occur within the same microenvironment, termed as a replication
fork. Responsible for DNA replication is DNA polymerase III, which catalyzes the
chemical reaction of DNA synthesis by creating phosphodiester bonds between
deoxyribonucleotides in a DNA chain. To achieve high speed replication, the highly
processive DNA polymerases utilize a similar strategy to replicate chromosomes in
bacteria, archaebacteria, and eukaryotes.

Processive DNA polymerases are assembled from three functional components: a

DNA polymerase/exonuclease, a ring-shaped sliding clamp, and a clamp loader. To
achieve processive and high speed replication of both the leading and lagging strands
in a coordinated manner, DNA polymerase utilizes sliding clamps to move along the
template without falling off. Sliding clamps are loaded onto DNA by specialized
protein complexes known as clamp loaders. Clamp loaders are ATP-fueled molecular
machines that open the sliding clamp, load then onto primed DNA, and unload them
at the appropriate time.

STRUCTURE
 E. coli Sliding Clamp
Eukaryotic Sliding Clamp

SLIDING CLAMP:
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 Nuclear Antigen.

E. coli T4 Bacteriophage
ß clamp, homodimer gp43, homotrimer of gene 43 prot

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
synthesis.
 Schematic representation of DNA:RFC:PCNA model

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 ATPases.

E. coli T4 Bacteriophage
gamma complex consisting of 5 subunits gene 44/62 protein complex consisting

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 three conserved 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.

FUNCTION
 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 the clamp.

E. coli clamp loading complex mechanism

EUKARYOTIC DNA CLAMP LOADING:

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.

Polymerase 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 control.

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)

References:

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.