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

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


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the template can be joined to make a long, continuous strand of DNA, the short RNA primers must be removed. This process is carried out by DNA polymerase I using its flap exonuclease activity to displace and cleave the RNA strand (Figure 1.9). As DNA polymerase I displaces the RNA primer, it extends the upstream (i.e., 5′) DNA that was previously polymerized by DNA polymerase III (Figure 1.8). Ribonuclease (RNase) H may contribute to this process under some circumstances by using its ability to degrade the RNA strand of a DNA-RNA double helix (Table 1.1). The Okazaki fragments are then joined together by DNA ligase as the replication fork moves on, as shown in Figure 1.8. By using RNA rather than DNA to prime the synthesis of new Okazaki fragments, the cell likely lowers the mistake rate of DNA replication (see below).

      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

      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


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