DNA Replication:- DNA Replication Requires Many Enzymes and Protein Factors
Replication in E. coli requires not just a single DNA polymerase but 20 or more different enzymes and proteins, each performing a specific task. The entire com plex has been termed the DNA replicase system or replisome. The enzymatic complexity of replication reflects the constraints imposed by the structure of DNA and by the requirements for accuracy. The main classes of replication enzymes are considered here in terms of the problems they overcome. Access to the DNA strands that are to act as templates requires separation of the two parent strands. This is generally accomplished by helicases, enzymes that move along the DNA and separate the strands, using chemical energy from ATP. Strand separation creates topological stress in the helical DNA structure , which is relieved by the action of topo isomerases. The separated strands are stabilized by DNA-binding proteins. As noted earlier, before DNA polymerases can begin synthesizing DNA, primers must be present on the template—generally short segments of RNA synthesized by enzymes known as primases. Ultimately, the RNA primers are removed and replaced by DNA; in E. coli, this is one of the many functions of DNA polymerase I. After an RNA primer is removed and the gap is filled in with DNA, a nick remains in the DNA backbone in the form of a broken phosphodiester bond. These nicks are sealed by DNA ligases. All these processes require coordination and regulation, an interplay best characterized in the E. coli system.
FIGURE 1 DNA polymerase III. (a) Architecture of bacterial DNA polymerase III. Two core domains, composed of subunits α, ᴇ, and Ө, are linked by a five-subunit γ complex (also known as the clamp-loading complex) with the composition ℸ2γδδ. The γ and ℸ subunits are encoded by the same gene. The subunit is a shortened version of ℸ; the ℸ subunit thus contains a domain identical to , along with an additional segment that interacts with the core polymerase. The other two subunits of DNA polymerase III*, and (not shown), also bind to the complex. Two clamps interact with the two-core subassembly, each clamp a dimer of the subunit. The complex interacts with the Dna B helicase through the subunit. (b) Two β subunits of E. coli polymerase III form a circular clamp that surrounds the DNA. The clamp slides along the DNA molecule, increasing the processivity of the polymerase III holoenzyme to greater than 500,000 by preventing its dissociation from the DNA. The end-on view shows the two β subunits as gray and light-blue ribbon structures surrounding a space-filling model of DNA. In the side view, surface contour models of the β subunits (gray) surround a stick representation of a DNA double helix (light and dark blue) (derived from PDB ID 2POL).
