Snyder and Champness Molecular Genetics of Bacteria. Tina M. Henkin

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.

Replication of Double-Stranded DNA

      Additional complications of DNA replication come from the fact that the DNA is double stranded and the strands are antiparallel. The replication of all bacterial chromosomes begins at one point, called the origin of replication, with the replication enzymes moving in opposite directions from this point along the chromosome. In this process, both strands of DNA are replicated at the same time with a coordinated set of proteins. Replicating the antiparallel strands is further complicated by the abovementioned fact that DNA polymerases can replicate only in the 5′-to-3′ direction. Therefore, one DNA strand is replicated in the same direction that the replication fork is moving, and in theory, replication of this strand could continue without the need for reinitiating in a process called leading-strand DNA synthesis. However, replication of the other DNA strand occurs in the opposite direction from the progression of the replication machinery. Replication of this strand must continually be reinitiated in a process known as lagging-strand DNA synthesis. Replication of double-stranded DNA requires coordination between multiple holoenzyme subunits and DNA polymerases, as well as a host of other replication proteins.

      SEPARATING THE TWO TEMPLATE DNA STRANDS

To serve as templates for DNA replication, the two DNA strands must be separated, a task that DNA polymerase cannot perform on its own. The strands must be separated because the bases of the DNA are inside the double helix, where they are not available to pair with the incoming deoxynucleotides to direct which nucleotide will be inserted at each step. Proteins called DNA helicases separate the strands of DNA (see Singleton et al., Suggested Reading). Many of these proteins form a ring around one strand of DNA and propel the strand through the ring, acting as a mechanical wedge that strips the strands apart as it moves. It takes a lot of energy to separate the strands of DNA, and helicases cleave a lot of ATP for energy, forming ADP in the process. There are about 20 different helicases in E. coli, and each helicase works in only one direction, either the 3′-to-5′ or the 5′-to-3′ direction. The DnaB helicase that normally separates the strands of DNA ahead of the replication fork in E. coli is a large doughnut-shaped complex composed of six polypeptide products of the dnaB gene. It propels one strand, the template for lagging-strand DNA replication, through the center of the complex in the 5′-to-3′ direction, opening strands of DNA ahead of the replication fork (Figure 1.10). The DnaB ring cannot load onto single-stranded DNA on its own to start a DNA replication fork; it requires the loading protein DnaC. Other helicases are discussed in later chapters in connection with recombination and repair.

      Once the strands of DNA have been separated, they also must be prevented from coming back together (or from annealing to themselves if they happen to be complementary over short regions). Separation of the strands is maintained by proteins called single-strand-binding (SSB) proteins or, less frequently, helix-destabilizing proteins. They are proteins that bind preferentially to singlestranded DNA and prevent double-stranded helical DNA from reforming prematurely. Interestingly, SSB activity goes beyond this passive role. SSB is also responsible for recruiting a number of replication and repair proteins through a specific set of amino acids encoded in the very C-terminal end of SSB, allowing it to serve as an organizational hub for other processes.

      PROCESSING THE TWO TEMPLATE DNA STRANDS

      As discussed above, the antiparallel configuration of DNA requires that the two DNA polymerases travel in two different directions while still allowing the larger replication machine to travel in one direction down the chromosome (Figure 1.8). This leads to fundamental differences in the natures of leading- and lagging-strand DNA replication. While replication of the leading-strand template can occur as soon as the strands are separated by the DnaB helicase, replication of the lagging-strand template is consistently reinitiated approximately every 1 to 2 kilobases (kb); this slows the process, hence the name lagging-strand synthesis. The short pieces of DNA produced from the lagging-strand template are called Okazaki fragments. Synthesis of each Okazaki fragment requires a new RNA primer about 10 to 12 nucleotides in length. In E. coli, these primers are synthesized by DnaG primase at the template sequence 3′-GTC-5′, beginning synthesis opposite the T. These RNA primers are then used to prime DNA synthesis by DNA polymerase III, which continues until it reaches the last RNA primer produced by DnaG (Figure 1.8). Before these