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Replication of large DNAs requires many functions that reside in proteins separate from the subunit used for polymerizing the chain of nucleotides. These functions include the coordination of multiple DNA polymerases and tethering of these components to the template DNA strands as a moving production platform. DNA polymerase III is the major DNA replication protein in E. coli responsible for polymerizing the new complementary DNA strands, and it functions with multiple DNA polymerase accessory proteins that travel along the template strand with the molecule of DNA polymerase III. The term DNA polymerase III holoenzyme can be used to describe the entire complex of proteins. The various subunits and subassemblies of the DNA polymerase III holoenzyme were originally identified from fractionation procedures and were designated by Greek letters (Table 1.1).

Figure 1.8 Discontinuous synthesis of one of the two strands of DNA during chromosome replication. (1) DNA polymerase (Pol) III replicates one strand, and the primase synthesizes RNA on the other strand in the opposite direction. (2) Pol III extends the RNA primer to synthesize an Okazaki fragment. (3) The primase synthesizes another RNA primer. (4) Pol III extends this primer until it reaches the previous primer. (5) Pol I removes the first RNA primer and replaces it with DNA. (6) DNA ligase seals the nick to make a continuous DNA strand, and the process continues. The strand that is synthesized continuously is the leading strand; the strand that is synthesized discontinuously is the lagging strand.

Figure 1.9 DNA polymerase I can remove an RNA primer by using strand displacement and endonuclease activity. (A) The DNA strand produced by DNA polymerase III holoenzyme is extended by DNA polymerase I until it encounters a previously synthesized RNA primer. (B) During the process of DNA replication, DNA polymerase I displaces the RNA primer. (C) An endonuclease activity in DNA polymerase I is used to cleave off the RNA primer. (D) Ligase joins the new Okazaki fragment to the previous Okazaki fragment to allow a contiguous DNA.

One of the DNA polymerase accessory proteins forms a ring around the template DNA strand and is responsible for keeping DNA polymerase from falling off. Because this ring slides freely over double-stranded DNA and will not easily come off the DNA, it is also referred to as a sliding clamp, or β clamp. The β clamp provides the foundation of the mobile platform for DNA replication, allowing it to continue for long distances without being released. In bacteria, the β clamp is a product of the dnaN gene, where two head-to-toe molecules form the ring around the DNA. While it was first isolated as part of the DNA polymerase III holoenzyme, the β clamp protein is important for multiple DNA transactions.

A special subcomplex within DNA polymerase III is the clamp loader, which is responsible for loading β clamp proteins onto the DNA. The clamp-loading complex is also responsible for tethering proteins across the DNA replication fork; the clamp loader binds the DNA polymerases on both DNA template strands and the enzyme responsible for separating the DNA strands (see below). The clamp loader is a complicated structure that consists of one γ and two τ proteins and one each of δ and δ', which form a five-sided structure, and two additional proteins, χ and ψ. The clamp loader complex is also responsible for removing β clamps. The rate of clamp removal allows many β clamps to reside on the DNA for a period of time after the replication fork passes (see Moolman et al., Suggested Reading). β clamps temporarily left behind on the newly replicated DNA play a role in helping to recruit other proteins responsible for various replication and repair functions described in this and other chapters.