Bacterial DNA Replication and Cell Division Help
Bacterial DNA Replication
The circular chromosome of bacteria presents special problems for replication. Circular chromosomes usually have a single site, called the origin, or ori, site, at which replication originates. By contrast, many ori sites exist on each chromosome of eukaryotes. Once the replication process starts, it usually proceeds bidirectionally from the ori site to form two replication forks.
As the two strands of a right-handed, double-helical, circular DNA unwind during replication, the molecule tends to become positively supercoiled or overwound, i.e., twisted in the same direction as the strands of the double helix. These supercoils are so tight that they would interfere with further replication if they were not removed. Topoisomerases are a group of enzymes that can change the topological or configurational shape of DNA. DNA gyrase is a bacterial topoisomerase that makes double-stranded cuts in the DNA, holds on to the broken ends so they cannot rotate, passes an intact segment of DNA through the break, and then reseals the break on the other side (Fig. 10-2).
Fig. 10-2. A proposed mechanism whereby DNA gyrase "pumps" negative supercoiling into DNA. A relaxed, covalently closed, circular DNA molecule (a) is bent into a configuration for strand passage (b). DNA gyrase makes double-stranded cuts (c), holds on to the ends, passes an intact segment through the break, and reseals the break on the other side (d).
This action of DNA gyrase quickly removes positive supercoils and momentarily relaxes the DNA molecule into a more energetically stable state. However, with the expenditure of energy, DNA gyrase normally pumps negative supercoiling or under winding (twisting in a direction opposite to the turns of the double helix) into relaxed DNA circles so that virtually all DNAs in both prokaryotes and eukaryotes naturally exist in the negative super-coiled state. Relaxed circles and positively super-coiled DNA exist only in the laboratory. Localized regions of DNA transiently and spontaneously unwind to single-stranded "bubbles" and then return to their former topology as hydrogen bonds between complementary base pairs are broken and reformed by thermal agitation. The strain of underwinding is thus momentarily relieved in a superhelix by an increase in the number, size, and duration of these bubbles. An equilibrium normally exists between these super-coiled and "bubbled" states. More bubbles form as the temperature increases.
At each replication fork, an enzyme called helicase unwinds the two DNA strands. Single-stranded, DNA binding (SSB) proteins protect the single-stranded regions in the replication forks from forming intrastrand base pairings that could cause a tangle of partially double-stranded segments that would interfere with replication. The enzyme primase synthesizes short RNA primers using a region on each strand as a template. Primers are required for DNA polymerase to begin extending the new DNA strand because DNA polymerase requires a 3'OH to initiate the bonding reaction.
Three DNA polymerase enzymes (referred to as pol I, pol II, and pol III) have been found in E. coli. Pol III is the principal replicating enzyme. Gaps left by pol III are filled by pol I, and DNA ligase seals the nicks. The function of pol II is not well established, although it is known that it is not involved in RNA primer replacement. In addition to their 5' to 3' synthetic activity, both pol I and pol III have 3' to 5' exonuclease activity, which plays a "proofreading" role by removing mismatched bases mistakenly inserted during chain polymerization. Pol I also has 5' to 3' exonuclease activity by which it normally removes primers and replaces them with complementary DNA sequences after polymerization has begun. About halfway through the above replication process, the replicative intermediate molecule looks like the Greek letter theta (θ), so is referred to as theta replication (Fig. 10-3).
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