Bacterial DNA Replication and Cell Division Help (page 2)
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).
Another type of bacterial replication is used to transfer a linear DNA molecule during bacterial conjugation or for the production of linear phage genomes. A nick occurs in one strand of a DNA double helix, creating free 3'-OH and 5'-P termini. Helicase and SSB proteins establish a replication fork. No primer is necessary because a strand with a free 3'-OH is available for elongation by DNA polymerase III as the leading strand. Simultaneously with replication of the leading strand, the template for the lagging strand is displaced. The displaced strand is discontinuously replicated to produce Okazaki fragments in the usual way (see Fig. 3-11).
The result of this replication model is a circle with a linear tail, resembling the Greek letter sigma (σ). Hence, this model is called sigma replication or rolling-circle replication (Fig. 10-4).
The circle may revolve several times, creating concatemers or covalently connected, linear repetitions of bacterial genomes. An endonuclease makes cuts at slightly different positions on each DNA strand of the concatemer to create genome-sized segments containing "sticky ends" (single-stranded complementary ends). The linear genomes circularize by base pairing of the sticky ends. DNA ligase seals each gap to create covalently closed (circular), double-stranded DNA molecules.
A replicating bacterial chromosome is thought to be attached to invaginations of the cell membrane at each replication fork. After DNA replication, the cell elongates by growth of the sector between the two attachment points, causing the two chromosomal replicas to move apart. A septum of new cell membrane and wall is then synthesized between the two chromosomes, creating two progeny cells (Fig. 10-5). The "passing down" of DNA from parent to progeny cell is called vertical gene transfer and the overall process of bacterial cell division is called binary fission.
Fig. 10-5. A model for segregation of bacterial DNA ("chromosome") replicas. (1) A circular DNA molecule is attached to invaginations of the cell membrane at two points. The DNA is a theta structure (about half replicated). (2) Replication is complete. (3) Cell division is beginning via growth of the membrane region shown in medium gray. Both daughter chromosomes are already partially replicated. (4) Cell division is complete. (5) Daughter cells are midway through the next generation. The membrane attachment points have moved to the center of the cell as a result of growth of new membrane (dark gray) after cell division. (After G. S. Stent and R. Calendar, Molecular Genetics, 2nd ed., W. H. Freeman and Company, New York, 1978.)
When bacteria are growing exponentially, most cells contain two to four identical chromosomes in various stages of replication. If a mutation occurs during replication, the new copy of DNA will be slightly different from the parental template. If this mutation occurs in a coding region that results in a defective protein, the effect of the mutation (i.e., the phenotype) will only be observable once the new cell has depleted its level of wild-type protein. This is a phenomenon known as phenotypic lag.
EXAMPLE 10.4 Resistance to a specific bacteriophage can be acquired by mutation of a gene responsible for the phage receptor on the cell's surface. Resistance cannot be fully realized until the receptor sites (synthesized under the direction of the former phage-sensitive genotype) have been completely diluted out through successive cell divisions. If even one receptor remains on the mutant cell, it is still susceptible to phage infection. Thus, many cell generations may be required before a phage-resistant mutation can be fully expressed in a progeny cell.
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