The chromosome of E. coli is reproduced through essentially continuous synthesis of the leading strand and discontinuous synthesis of discrete Okazaki fragments on the lagging strand. Following that, the Okazaki fragments are processed and ligated into high molecular weight DNA (Ogawa and Okazaki 1980, Kornberg and Baker 1992). The DNA polymerase III holoenzyme (holoenzyme) is made up of two pol III cores linked together by a τ dimer. This connection mechanism τ tethers the lagging strand polymerase to the fork, allowing it to be recycled during the subsequent rounds of Okazaki fragment synthesis.
Furthermore, adding ATPγS to isolated complexes produced in the presence of ATP causes one-half of the initiation complexes to lose activity. Once the ATP binding site of DnaX is saturated, the 2-fold differential nucleotide phenomenon is not an equilibrium artifact because final activity is independent of ATPγS concentration. Over time, reactions did not exceed 50%. Traditionally, the functional asymmetry of holoenzyme has been blamed for this unequal nucleotide impact.
Structure
DNA polymerase III is a holoenzyme that consists of two core enzymes, each with three subunits (α, ɛ and θ) , a sliding clamp with two beta subunits, and a clamp-loading complex with several subunits (δ, τ, γ, ψ, and χ).
The replisome is made up of the following components:
- There are two DNA Pol III enzymes, each with subunits, and. (It has been established that Pol III possesses a third copy at the replisome.)
- The polymerase activity is controlled by the α subunit (encoded by the dnaE gene).
- 3’→5′ exonuclease activity is present in the ε subunit (dnaQ).
- The θ subunit (holE) induces the proofreading of the subunit.
- The polymerase is kept attached to the DNA by two β units (dnaN) that act as sliding DNA clamps.
- 2 τ units (dnaX) that work together to dimerize two of the key enzymes (α, ε, and θ subunits).
- 1 γ unit (also dnaX) that helps the two β subunits form a unit and attach to DNA by acting as a clamp loader for the lagging strand Okazaki fragments. The unit consists of five γ subunits: three γ subunits, one δ subunit (holA), and one’ δ’ subunit (holB). The δ is involved in the lagging strand’s copying.
- X (holC) and Ψ (holD), which bind to γ or τ and create a 1:1 complex. The transition from RNA primer to DNA can also be mediated by X.
Function
The third polymerase, Pol III, is primarily responsible for chromosomal DNA duplication, whereas other DNA polymerases are mostly responsible for DNA repair and translesion DNA synthesis. Pol III HE is part of the replicative apparatus that operates at the replication fork, together with a DNA helicase and a primase. Pol III is a multi-subunit complex, unlike other bacterial DNA polymerases, in which twin catalytic subassemblies, known as the Pol III core, are embedded with several other auxiliary subunits. These subunits work together to allow Pol III to function as a chromosomal replicase, manufacturing both the leading and lagging strands of DNA at the same time. Pol III HE DNA synthesis is further distinguished by a quick chain-elongation reaction, high processivity, and high fidelity, all of which are required for chromosomal DNA replication.
CONCLUSION
In an ATP-dependent process, the DNA Polymerase III holoenzyme generates initiation complexes on primed DNA. We show that ATPS, a nonhydrolyzable ATP analogue, promotes the development of an isolable leading strand complex that only loads and repeats the lagging strand in the presence of ATP and the single-stranded DNA binding protein. By an orderly mechanism, the single endogenous DnaX complex within DNA polymerase III holoenzyme assembles onto both the leading and lagging strand polymerases. The dimeric replication complex disassembles in the reverse order in which it was constructed. These findings prove that holoenzyme is a symmetric dimer with distinct leading and trailing strand polymerases. Over the last decade, researchers have made significant progress in understanding the characteristics of a true replicative complex by studying the structure, function, and control of this enzyme. At least seven distinct subunits make up the holoenzyme. The catalytic core is made up of three of these molecules: alpha, epsilon, and theta. The catalytic subunit alpha appears to be the product of the dnaE gene.
Theta subunit’s function is still unknown. On natural chromosomes, the polymerase requires the auxiliary subunits beta, gamma, and delta, which are encoded by dnaN, dnaZ, and dnaX, respectively. All of the proteins help the polymerase become more processive and create an initiating complex in the presence of ATP. Tau causes the polymerase to dimerize, possibly generating a structure that allows leading and lagging strand synthesis to be coordinated at the replication fork. This dimeric compound could be asymmetric, with characteristics that match the requirements for strand synthesis in the leading and lagging directions.