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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 protein–protein 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 α+β 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 γ,δ,δ',χ,ψ 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 αε polymerase complex.
Dr. Shiva C. Aithal, Dept. of Microbiology, Dnyanopasak College, PARBHANI (Maharashtra) INDIA
beta Dr. The α subunit possesses DNA polymerase activity and the ε subunit is a 3’-5’ exonuclease. Shiva C. The beta chain of bacterial DNA polymerase III is composed of three topologically nonequivalent domains (N-terminal. Dept. Dnyanopasak College. Two beta chain molecules are tightly associated to form a closed ring encircling duplex DNA. central. 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. gamma complex (clamp loader). 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.000 grams per mole). It is also referred to as polC. but cannot slide on single-stranded DNA or single-stranded DNA coated with SSB. The proteins (called subunits) that associate with pol III in the holoenzyme perform several functions. subassemblies. but it can be loaded onto nicked circles. By acting as a sliding "clamp". gapped circles. or single-stranded circular DNA. However. This multi-protein complex is referred to as the pol III holoenzyme. uses ATP energy when loading beta onto primed DNA. covalently closed circular DNA. coli is a single protein of molecular weight 130 kDa (130. Aithal. Though the molecule has DNA polymerase activity by itself. The beta subunit can be loaded onto DNA by the clamp loader (gamma complex) in an ATPdependent reaction). that is. (The clamp loader also unloads clamps!) Beta cannot be loaded onto linear DNA . The Table summarizes the pol III subunits. PARBHANI (Maharashtra) INDIA Page 2 . linearization of the nicked circle with a restriction endonuclease releases beta from the DNA. It can slide along double-stranded DNA (or DNA-RNA in double-stranded form). of Microbiology. Once loaded onto a nicked circle. The most interesting subunit is called beta. and primed single-stranded circles. dnaE. DNA Polymerase III (pol III) from E. and C-terminal). polIII works to replicate DNA in the bacterial cell in conjunction with other proteins. beta is a sliding clamp. beta stays associated with the DNA. which forms a donut shaped ring around the DNA and helps to anchor the holoenzyme to the DNA during replication. or the alpha subunit. clamp loader requires and recognizes a 3'-hydroxylterminus (primer-terminus). that is. Pol III holoenzyme directs both leading and lagging strand synthesis simultaneously by virtue of having two polymerase subunits. beta helps the holoenzyme to replicate long stretches of DNA without "falling off" the strand (this is called processivity).
Can only isolate exonuclease (mutants lacking this Activities conditional-lethal dnaE mutants. Processivity the fork until replication terminates. e. "filling in" DNA to replace the short (about 10 nucleotides) RNA primers on Okazaki fragments.Quick Comparison of DNA polymerases I and III DNA polymerase III DNA polymerase I DNA Pol III holoenzyme is an asymmetric dimer. and 5'-to-3' polymerase in the cell. Synthesizes essential activity are not viable). primers on the lagging strand. Shiva C. It is there are 6 forks. but disassociates after each RNA primer is removed. 250-1. reassociate with the lagging strand easily. without beta. PARBHANI (Maharashtra) INDIA Page 3 . This is the replicative exonuclease. so having 5'-to-3' Structure leading and lagging strands. 10-20 molecules/cell.. then only 12 core concentration means that it can polymerases are needed for replication. If one processive holoenzyme distributive. Dnyanopasak College. About 400 molecules/cell. DNA polymerase III holoenzyme (Note: no beta subunits are shown. DNA Pol I is a monomeric protein i. The beta subunit is a sliding Distributive. so the higher Molecules/cell (two cores) is at each fork. both leading and lagging strands. this form of the complex is called DNA pol III) Dr. Aithal. Pol I does NOT remain clamp. but is capable of Vmax (nuc. and fill-in the resulting gaps. This is NOT the rate of replication measured in Cairns' fast enough to be the main experiments. Polymerization and 3'-to-5' exonuclease. Only this polymerase is fast replicative enzyme. Highly processive. The holoenzyme remains associated with associated with the lagging strand. two cores with other accessory subunits. but on Polymerization. In rapidly growing cells. 3'-to-5' different subunits. of Microbiology. It is can thus move with the fork and make both distributive./sec) enough to be the main replicative enzyme. Dept. This is as fast as 20 nucleotides/second. exonuclease and polymerase on the same molecule for removing RNA primers is effective and efficient. It with three active sites. Primary function is to remove RNA No 5' to 3' exonuclease activity.000 nucleotides/second.
The beta subunit. The sliding clamps and their associated clamp loading systems are of broad importance in many cellular processes involving DNA. Once attached to the beta subunit. incorporating new nucleotides into the growing DNA strand at speeds as high as 750 nucleotides per second. Dnyanopasak College. PARBHANI (Maharashtra) INDIA Page 4 .Steps involved in loading a sliding clamp for processive DNA synthesis. Shiva C. Dept. known as a sliding clamp. Aithal. of Microbiology. Dr. 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 catalytic alpha subunit of the polymerase can move along DNA for tens of kilobases or more without dissociation. beyond that originally imagined by their discovery as essential factors for chromosomal replication. forms a stable ring-shaped structure that encircles DNA.
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