What actually happens at the replication fork is more complicated than is suggested by the simple picture given so far. For one thing, this picture ignores the overall topological restraints on the replicating DNA. The topology of a molecule refers to its position in space. Because the circular DNA is very long and its strands are wrapped around each other, pulling the two strands apart introduces stress into other regions of the DNA in the form of supercoiling. If no mechanism existed to allow the two strands of DNA to rotate around each other, supercoiling would cause the chromosome to look like a telephone cord wound up on itself, an event that has been experimentally shown to eventually halt progression of the DNA replication fork. To relieve this stress, enzymes called topoisomerases work to help undo the supercoiling ahead of the replication fork. DNA supercoiling and topoisomerases are discussed below. The fork itself can also twist when the supercoiling that builds up ahead of the replication fork diffuses behind the replication fork, a process that twists the two new strands around one another and that is also sorted out by topoisomerases (see below).
COORDINATING REPLICATION OF THE TWO TEMPLATE STRANDS
The picture of the two strands of DNA replicating independently, as shown in Figure 1.8, does not take into consideration all of the coordination that must occur during DNA replication. The anatomy of the larger complex of replication factors remains unresolved; however, interactions among many of these components provide a hint as to how the larger complex functions (Figure 1.10). Rather than replicating independently, the DNA polymerases that produce the leading-strand and lagging-strand DNAs are joined to each other through the τ subunits of the holoenzyme (Table 1.1). In the holoenzyme there are two τ subunits and a derivative product called γ, which is incapable of interacting with DNA polymerase. The γ and τ subunits are encoded by the same gene, dnaX. Expression of the full gene results in production of the longer τ subunit, whereas a stutter in how the protein is produced from this gene, called a “frameshift,” produces the shorter γ product. The configuration of having two τ and γ subunits may be important to ensure that only two DNA polymerase III molecules are at the replication fork, possibly facilitating the use of alternate polymerases for repair when needed (see below and Dohrmann et al., Suggested Reading).
Figure 1.10 “Trombone” model for how both the leading strand and lagging strand might be simultaneously replicated at the replication fork. RNA primers are shown in green, and their initiation sites are shown as the sequence 3′-GTC-5′ boxed in blue. (A) The Pol III holoenzyme synthesizes lagging-strand DNA initiated from priming site 2 and runs into the primer at site 1. (B) The DNA strand undergoing lagging-strand replication loops out of the replication complex as the leading-strand polymerase progresses and the lagging-strand polymerase replicates toward the last Okazaki fragment. (C) Pol III has been released from the laggingstrand template at priming site 1 and has hopped ahead, leaving the old β clamps behind, and has reassembled with a new β clamp on the DNA at primer site 3 to synthesize an Okazaki fragment. Both the leading-strand and lagging-strand Pol III enzymes remain bound to each other and the helicase through interactions with τ during the release and reassembly process. (D) Pol III continues synthesis of the lagging strand from priming site 3 while Pol I is removing the primer at site 1 and replacing it with DNA. The Pol III holoenzyme hops to the primer at site 4 after reaching the primer at site 2. The primers and Okazaki fragments are not drawn to scale.
To accommodate the fact that the two DNA polymerases must move in opposite directions and still remain tethered, the lagging-strand template probably loops out as an Okazaki fragment is synthesized. The loop is then relaxed as the polymerase on the lagging-strand template is released from the β clamp, allowing the DNA polymerase to rapidly and efficiently “hop” ahead to the next RNA primer to begin synthesizing the next Okazaki fragment (Figure 1.10). The polymerase associates with a new β clamp assembled by the clamp loader at the site of the new RNA primer, while the old β clamp is left behind. The β clamp left on the last Okazaki fragment plays important roles in finishing synthesis and joining the fragments of lagging-strand DNA via interactions with DNA polymerase I, ligase, and repair proteins. β clamps are eventually recycled, possibly through the removal function of the δ subunit of the clamp loader. This model involving the looping out of the lagging-strand template has been referred to as the “trombone” model of replication because the loops forming and contracting at the replication fork resemble the extension and return of the slide of the musical instrument. The situation is probably similar in all bacteria and even the other domains of life, although in some other bacteria, including Bacillus subtilis, and in eukaryotes, different combinations of DNA polymerases are used to polymerize the leading and lagging strands (see Sanders et al., Suggested Reading). In the case of B. subtilis, an additional DNA polymerase interacts with DnaG and the replicative helicase, extending the RNA primer with DNA before handing the template off to the DNA polymerase used for the majority of DNA replication on both strands.
In addition to its role in loading β clamps onto template DNA, the clamp loader also plays an important role in coordinating the various replication components. Not only does the τ subunit of the clamp loader interact with DNA polymerase on the leading-strand and laggingstrand templates, it also interacts with the DnaB helicase (Figure 1.10). Further coordination on the lagging-strand template is facilitated by interactions between the DnaG primase and DnaB helicase (Figure 1.10). Coordination through the clamp loader helps to focus the energy from DNA polymerization with the energy that powers the helicase, allowing a high rate of DNA replication. The interaction between DNA polymerase III and DnaB governs the speed of unwinding so that it matches the rate of DNA polymerization to prevent undue exposure of singlestranded DNA.
THE GENES FOR REPLICATION PROTEINS
Most of the genes for replication proteins have been found by isolating mutants defective in DNA replication, but not RNA or protein synthesis. Since a mutant cell that cannot replicate its DNA will die, any mutation (for definitions of mutants