BETA CLAMP AND PROGRESSIVE POLYMERASES

A DNA clamp, also known as a sliding clamp, is a protein fold that serves as a processivity-promoting factor in DNA replication. As a critical component of the DNA polymerase III holoenzyme, the clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand. The clamp-polymerase proteinprotein interactions are stronger and more specific than the direct interactions between the polymerase and the template DNA strand; because the rate-limiting step in the DNA synthesis reaction is the association of the polymerase with the DNA template, the presence of the sliding clamp dramatically increases the number of nucleotides that the polymerase can add to the growing strand per association event. The presence of the DNA clamp can increase the rate of DNA synthesis up to 1,000-fold compared with a nonprocessive polymerase. STRUCTURE The DNA clamp fold is an + protein that assembles into a multimeric structure that completely encircles the DNA double helix as the polymerase adds nucleotides to the growing strand. The DNA clamp assembles on the DNA at the replication fork and "slides" along the DNA with the advancing polymerase, aided by a layer of water molecules in the central pore of the clamp between the DNA and the protein surface. Because of the toroidal shape of the assembled multimer, the clamp cannot dissociate from the template strand without also dissociating into monomers. The first indication for the toroid shape of the sliding clamps came from the study of the b subunit of the E. coli replicase. The DNA clamp fold is found in bacteria, archaea, eukaryotes and some viruses. In bacteria, the sliding clamp is a homodimer composed of two identical beta subunits of DNA polymerase III and hence is referred to as the beta clamp. In archaea and eukaryotes, it is a trimer composed of three molecules. The T4 bacteriophage also uses a sliding clamp, called gp45. BACTERIAL BETA CLAMP The beta clamp is a specific DNA clamp and a subunit of the DNA polymerase III holoenzyme found in bacteria. The -subunit clamp ensures that the polymerase stays on the DNA Two beta subunits are assembled around the DNA by the gamma subunit and ATP hydrolysis; this assembly is called the pre-initiation complex. After assembly around the DNA, the beta subunits' affinity for the gamma subunit is replaced by an affinity for the alpha and epsilon subunits, which together create the complete holoenzyme. DNA polymerase III is the primary enzyme complex involved in prokaryotic DNA replication. The gamma complex of DNA polymerase III, composed of ,,',, subunits, catalyzes ATP to chaperone two beta subunits to bind to DNA. Once bound to DNA, the beta subunits can freely slide along double stranded DNA. The beta subunits in turn bind the polymerase complex.
Dr. Shiva C. Aithal, Dept. of Microbiology, Dnyanopasak College, PARBHANI (Maharashtra) INDIA

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The subunit possesses DNA polymerase activity and the subunit is a 3-5 exonuclease. The beta chain of bacterial DNA polymerase III is composed of three topologically nonequivalent domains (N-terminal, central, and C-terminal). Two beta chain molecules are tightly associated to form a closed ring encircling duplex DNA. DNA Polymerase III (pol III) from E. coli is a single protein of molecular weight 130 kDa (130,000 grams per mole). It is also referred to as polC, dnaE, or the alpha subunit. Though the molecule has DNA polymerase activity by itself, polIII works to replicate DNA in the bacterial cell in conjunction with other proteins. This multi-protein complex is referred to as the pol III holoenzyme. The proteins (called subunits) that associate with pol III in the holoenzyme perform several functions. The most interesting subunit is called beta, which forms a donut shaped ring around the DNA and helps to anchor the holoenzyme to the DNA during replication. By acting as a sliding "clamp", beta helps the holoenzyme to replicate long stretches of DNA without "falling off" the strand (this is called processivity). Pol III holoenzyme directs both leading and lagging strand synthesis simultaneously by virtue of having two polymerase subunits. The Table summarizes the pol III subunits, subassemblies, and their functions:
DNA polymerase III subunits and subassemblies Function Subassembly (complex) DNA polymerase 3'-to-5' exonuclease (editing exonuclease) stimulates 3'-to-5' exonuclease dimerizes cores, activates DnaB helicase activity binds ATP unknown core (there are two cores per DNA polymerase III holoenzyme)

Subunit alpha epsilon theta tau gamma delta

delta prime stimulates clamp loading chi psi interacts with SSB to allow removal of DnaG primase from primer unknown The Sliding clamp. The beta subunit can be loaded onto DNA by the clamp loader (gamma complex) in an ATPdependent reaction). (The clamp loader also unloads clamps!) Beta cannot be loaded onto linear DNA , covalently closed circular DNA, or single-stranded circular DNA, but it can be loaded onto nicked circles, gapped circles, and primed single-stranded circles; that is, clamp loader requires and recognizes a 3'-hydroxylterminus (primer-terminus). Once loaded onto a nicked circle, beta stays associated with the DNA. However, linearization of the nicked circle with a restriction endonuclease releases beta from the DNA; that is, beta is a sliding clamp. It can slide along double-stranded DNA (or DNA-RNA in double-stranded form), but cannot slide on single-stranded DNA or single-stranded DNA coated with SSB.

gamma complex (clamp loader), uses ATP energy when loading beta onto primed DNA.

beta

Dr. Shiva C. Aithal, Dept. of Microbiology, Dnyanopasak College, PARBHANI (Maharashtra) INDIA

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Quick Comparison of DNA polymerases I and III DNA polymerase III DNA polymerase I DNA Pol III holoenzyme is an asymmetric dimer; DNA Pol I is a monomeric protein i. e., two cores with other accessory subunits. It with three active sites. It is can thus move with the fork and make both distributive, so having 5'-to-3' Structure leading and lagging strands. exonuclease and polymerase on the same molecule for removing RNA primers is effective and efficient. Polymerization and 3'-to-5' exonuclease, but on Polymerization, 3'-to-5' different subunits. This is the replicative exonuclease, and 5'-to-3' polymerase in the cell. Can only isolate exonuclease (mutants lacking this Activities conditional-lethal dnaE mutants. Synthesizes essential activity are not viable). both leading and lagging strands. Primary function is to remove RNA No 5' to 3' exonuclease activity. primers on the lagging strand, and fill-in the resulting gaps. 250-1,000 nucleotides/second. This is as fast as 20 nucleotides/second. This is NOT the rate of replication measured in Cairns' fast enough to be the main experiments. Only this polymerase is fast replicative enzyme, but is capable of Vmax (nuc./sec) enough to be the main replicative enzyme. "filling in" DNA to replace the short (about 10 nucleotides) RNA primers on Okazaki fragments. Highly processive. The beta subunit is a sliding Distributive. Pol I does NOT remain clamp. The holoenzyme remains associated with associated with the lagging strand, Processivity the fork until replication terminates. but disassociates after each RNA primer is removed. 10-20 molecules/cell. In rapidly growing cells, About 400 molecules/cell. It is there are 6 forks. If one processive holoenzyme distributive, so the higher Molecules/cell (two cores) is at each fork, then only 12 core concentration means that it can polymerases are needed for replication. reassociate with the lagging strand easily.

DNA polymerase III holoenzyme (Note: no beta subunits are shown; without beta, this form of the complex is called DNA pol III)

Dr. Shiva C. Aithal, Dept. of Microbiology, Dnyanopasak College, PARBHANI (Maharashtra) INDIA

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Steps involved in loading a sliding clamp for processive DNA synthesis.

The beta subunit of DNA polymerase-III holoenzyme confers upon the polymerase the ability to faithfully track the rapidly moving replication fork while synthesizing leading and lagging strand DNA simultaneously. The beta subunit, known as a sliding clamp, forms a stable ring-shaped structure that encircles DNA. Once attached to the beta subunit, the catalytic alpha subunit of the polymerase can move along DNA for tens of kilobases or more without dissociation, incorporating new nucleotides into the growing DNA strand at speeds as high as 750 nucleotides per second. The sliding clamps and their associated clamp loading systems are of broad importance in many cellular processes involving DNA, beyond that originally imagined by their discovery as essential factors for chromosomal replication.

Dr. Shiva C. Aithal, Dept. of Microbiology, Dnyanopasak College, PARBHANI (Maharashtra) INDIA

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