Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies
This sequence arrangement is useful for two reasons. First, synthesis of the two strands can be measured separately: about 95% of label from radioactive dGTP goes into the leading strand, while 95% of label from radioactive dCTP goes into the lagging strand. Second, synthesis on the two strands can be specifically inhibited with different chain-terminating nucleotides. For example, dideoxy CTP (ddCTP; structure shown in panel C) would be incorporated 20-fold more frequently into the lagging-strand product than the leading-strand product, and thereby preferentially inhibit lagging-strand synthesis.
In this experiment, DNA replication is monitored with either radioactive dCTP (panel D, left graph; lagging-strand synthesis) or radioactive dGTP (right graph; leading-strand synthesis). After 2.5 minutes of the reaction, ddCTP (non-radioactive) is added to inhibit lagging-strand synthesis. Note that the chain-terminating nucleotide is added at an 80-fold lower concentration than normal dCTP, and so the chain terminator is only incorporated in a small fraction of extensions opposite dG residues. The striking result is that ddCTP addition rapidly inhibits both leading- and lagging-strand synthesis at roughly equal efficiencies. Evidently, the leading-strand polymerase is somehow induced to stop synthesis when its lagging-strand partner in the replisome is obstructed.
Panels A and D in this Figure were reproduced from Lee et al. (1998), with permission from Elsevier; permission conveyed by Copyright Clearance Center, Inc.
1Bacterial thioredoxin is also involved in assembly of certain bacteriophages, and again, the redox function of the protein is not required. Thioredoxin has been found to bind some 80 different bacterial proteins, suggesting that it may be involved as a structural component in additional processes.
2Thioredoxin also plays a key role here, configuring the DNA polymerase so it can bind the second site on the helicase/primase.
3These names are arbitrary, in that the six subunits are identical, but help in explaining the subunit behavior during helicase movement along the DNA.
4The third DNA polymerase visualized in these studies is bound to a different site in the helicase/primase complex than the secondary site discussed in Section 2.3; thus, a total of at least four potential DNA polymerase binding sites are involved in replication of T7 DNA.
5A number of animated videos of the trombone model can be found online, including those at www.youtube.com.
6Called gp6 for the aficionado.
7Called gp1.3.
Chapter 3
The highly efficient replication system of bacteria
DNA replication in both prokaryotic and eukaryotic cells is significantly more complex than in bacteriophage T7, and yet the basic functions and enzymatic mechanisms are very similar. In addition, the structural architecture of many of the key replication proteins, such as DNA polymerases and helicases, are analogous and sometimes evolutionarily related. The distinctions evident in cellular systems include the use of many more protein subunits in the overall replication reaction. One such difference of great importance is the use of specialized proteins, called sliding clamps, which tether cellular replicative polymerases to their DNA template and orchestrate polymerase behavior.
The most extensively studied cellular replication system is that of the bacterium, Escherichia coli, and this remarkably efficient system will be the focus of this chapter. Under optimal conditions, E. coli can duplicate its 4.6-million base-pair chromosome with high accuracy at a speed that allows the cells to divide every 20 minutes. Many bacterial species have replication proteins that are homologous to those of E. coli and presumably replicate by similar mechanisms, with only minor variations. However, it is worth noting that more distantly related bacteria sometimes show significant deviations from the E. coli model. For example, certain grampositive bacterial species such as Bacillus subtilis have an extra replicative polymerase with distinct properties. Without going into details, one of the Bacillus subtilis replicative polymerases is thought to extend the RNA primer on the lagging strand for a short distance, and then hand off the reaction to the other replicative polymerase (which also replicates the leading strand). As we will see in the next chapter, this kind of handoff is also a key feature of eukaryotic DNA replication.
3.1The E. coli replisome from 30,000 feet
To a first approximation, the E. coli chromosome is replicated in its entirety by just two replisome complexes loaded at a single origin site on the chromosome (also see Chapter 5). These two replisomes traverse the circular chromosome in opposite directions at a speed of roughly 1000 base pairs per second, and meet up to complete replication in a special terminus region on the opposite side of the circle. This speed, while quite impressive, is not quite high enough to explain how the bacterial cell can divide every 20 minutes (1000 base pairs/second × 60 seconds/minute × 20 minutes/division cycle × 2 replication forks = 2.4 million base pairs, only about half the number in the genome). This riddle is solved by the fact that E. coli and other bacteria actually carry out multiple rounds of replication at the same time under conditions of rapid cell division. Thus, two or three replisomes may be following each other around the chromosome at any one time, and partially replicated chromosomes must be segregated to daughter cells at the time of cell division (see Chapter 5).
A key question about DNA replication in the context of a living cell is whether the replisome is motoring along a relatively fixed DNA molecule or whether the DNA is slithering through a relatively fixed replisome. In the latter case, the DNA could actually be transported or “pumped” from one region of the cell to another during the act of replication. Scientists have been able to approach this question by placing visible markers (fluorophores) on the E. coli replisome proteins or, in a different experiment, particular locations of the chromosomal DNA. Strikingly, the replication proteins tended to remain near the middle of the cell throughout the period of DNA replication, while specific locations of the chromosome migrated in the cell. As replication progressed, the two duplicated copies of any particular chromosomal region moved in opposite directions from near the center of the cell toward the two poles. In this way, the movement of the duplicated chromosomes in preparation for bacterial cell division is coordinated with the act of replication. The separation of the two duplicated copies of the chromosome is made very challenging by the fact that each duplicated copy contains one of the two strands of parental DNA, and these two parental strands were wrapped around each other once every 10 or so base pairs before replication occurred. We will return to these topological issues in Chapter 7.
3.2The replicative DNA polymerase holoenzyme
Two different DNA polymerases are involved in E. coli DNA replication. For historical reasons, the main replicative polymerase for both leading- and lagging-strand synthesis is called DNA polymerase III, while the polymerase involved in processing Okazaki fragments is DNA polymerase I. DNA polymerase III can be isolated as a multi-protein complex called the “holoenzyme,” and the composition of the holoenzyme reveals much about the overall replisome (Figure 3.1A, right). The complex contains two copies of the actual DNA polymerase, which is a three-subunit enzyme that is referred to as the DNA polymerase III “core” (Figure 3.1A, left). In the context of chromosomal replication, one of these core enzymes replicates the leading strand and the other replicates the lagging strand.1 The holoenzyme also contains one copy of a multi-subunit complex that can load sliding clamps on the DNA, not surprisingly called the “clamp loader.” As we will see below, repetitive clamp loading is a central feature of lagging-strand