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	Eukaryotes
ß clamp, homodimer	gp43, homotrimer of gene 43 protein	PCNA, homotrimer

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	Eukaryotes
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 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.

DNA replication fork

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.