DNA Replication:- Replication of the E. coli Chromosome Proceeds in Stages
The synthesis of a DNA molecule can be divided into three stages: initiation, elongation, and termination, distinguished both by the reactions taking place and by the enzymes required. As you will find here and in the next two chapters, synthesis of the major information containing biological polymers—DNAs, RNAs, and proteins—can be understood in terms of these same three stages, with the stages of each pathway having unique characteristics. The events described below reflect in formation derived primarily from in vitro experiments using purified E. coli proteins, although the principles are highly conserved in all replication systems.
The E. coli replication origin, oriC, consists of 245 bp; it bears DNA sequence elements that are highly conserved among bacterial replication origins. The general arrangement of the conserved sequences is illustrated in Figure 1. The key sequences of interest here are two series of short repeats: three repeats of a 13 bp sequence and four repeats of a 9 bp sequence. At least nine different enzymes or proteins (summarized in Table 25–3) participate in the initiation phase of replication. They open the DNA helix at the origin and establish a prepriming complex for subsequent re actions. The crucial component in the initiation process is the DnaA protein. A single complex of four to five DnaA protein molecules binds to the four 9 bp repeats in the origin (Fig. 2, step 1), then recognizes and successively denatures the DNA in the region of the three 13 bp repeats, which are rich in A=T pairs (step 2). This process requires ATP and the bacterial histonelike protein HU. The DnaC protein then loads the DnaB protein onto the unwound region. Two ring shaped hexamers of DnaB, one loaded onto each DNA strand, act as helicases, unwinding the DNA bidirectionally and creating two potential replication forks. If the E. coli single-stranded DNA–binding protein (SSB) and DNA gyrase (DNA topoisomerase II) are now added in vitro, thousands of base pairs are rapidly unwound by the DnaB helicase, proceeding out from the origin. Many molecules of SSB bind cooperatively to single stranded DNA, stabilizing the separated strands and preventing renaturation while gyrase relieves the topo logical stress produced by the DnaB helicase. When additional replication proteins are included in the in vitro system, the DNA unwinding mediated by DnaB is coupled to replication, as described below. Initiation is the only phase of DNA replication that is known to be regulated, and it is regulated such that replication occurs only once in each cell cycle. The mechanism of regulation is not yet well understood, but genetic and biochemical studies have provided a few insights. The timing of replication initiation is affected by DNA methylation and interactions with the bacterial plasma membrane. The oriC DNA is methylated by the Dam methylase (Table 25–3), which methylates the N6 position of adenine within the palindromic sequence (5) GATC. (Dam is not a biochemical expletive; it stands for DNA adenine methylation.) The oriC region of E. coli is highly enriched in GATC sequences—it has 11 of them in its 245 bp, whereas the average frequency of GATC in the E. coli chromosome as a whole is 1 in 256 bp.
FIGURE 1 Arrangement of sequences in the E. coli replication origin, oriC. Although the repeated sequences (shaded in color) are not identical, certain nucleotides are particularly common in each position, forming a consensus sequence. In positions where there is no consensus, N represents any of the four nucleotides. The arrows indicate the orientations of the nucleotide sequences.
FIGURE 2 Model for initiation of replication at the E. coli ori gin, oriC. 1 About 20 DnaA protein molecules, each with a bound ATP, bind at the four 9 bp repeats. The DNA is wrapped around this complex. 2 The three A=T-rich 13 bp repeats are denatured sequentially. 3 Hexamers of the DnaB protein bind to each strand, with the aid of DnaC protein. The DnaB helicase activity further un winds the DNA in preparation for priming and DNA synthesis.
Immediately after replication, the DNA is hemi methylated: the parent strands have methylated oriC sequences but the newly synthesized strands do not. The hemimethylated oriC sequences are now sequestered for a period by interaction with the plasma membrane (the mechanism is unknown). After a time, oriC is released from the plasma membrane, and it must be fully methylated by Dam methylase before it can again bind DnaA. Regulation of initiation also involves the slow hydrolysis of ATP by DnaA protein, which cycles the protein between active (with bound ATP) and inactive (with bound ADP) forms on a timescale of 20 to 40 minutes.
The elongation phase of replication includes two distinct but related operations: leading strand syn thesis and lagging strand synthesis. Several enzymes at the replication fork are important to the synthesis of both strands. Parent DNA is first unwound by DNA helicases, and the resulting topological stress is relieved by topo isomerases. Each separated strand is then stabilized by SSB. From this point, synthesis of leading and lagging strands is sharply different. Leading strand synthesis, the more straightforward of the two, begins with the synthesis by primase (DnaG protein) of a short (10 to 60 nucleotide) RNA primer at the replication origin. Deoxyribonucleotides are added to this primer by DNA polymerase III. Leading strand synthesis then proceeds continuously, keeping pace with the unwinding of DNA at the replication fork. Lagging strand synthesis, as we have noted, is ac complished in short Okazaki fragments. First, an RNA primer is synthesized by primase and, as in leading strand synthesis, DNA polymerase III binds to the RNA primer and adds deoxyribonucleotides (Fig. 3). On this level, the synthesis of each Okazaki fragment seems straightforward, but the reality is quite complex. The complexity lies in the coordination of leading and lagging strand synthesis: both strands are produced by a single asymmetric DNA polymerase III dimer, which is accomplished by looping the DNA of the lagging strand as shown in Figure 25–14, bringing together the two points of polymerization.
FIGURE 3 Synthesis of Okazaki fragments. (a) At intervals, primase synthesizes an RNA primer for a new Okazaki fragment. Note that if we consider the two template strands as lying side by side, lagging strand synthesis formally proceeds in the opposite direction from fork movement. (b) Each primer is extended by DNA polymerase III. (c) DNA synthesis continues until the fragment extends as far as the primer of the previously added Okazaki fragment. A new primer is synthesized near the replication fork to begin the process again.
(Fig. 4c) The synthesis of Okazaki fragments on the lagging strand entails some elegant enzymatic choreography. The DnaB helicase and DnaG primase constitute a func tional unit within the replication complex, the primo some. DNA polymerase III uses one set of its core sub units (the core polymerase) to synthesize the leading strand continuously, while the other set of core subunits cycles from one Okazaki fragment to the next on the looped lagging strand. The DnaB helicase unwinds the DNA at the replication fork (Fig. 4a) as it travels along the lagging strand template in the 5n3 direction. DNA primase occasionally associates with DnaB helicase and synthesizes a short RNA primer (Fig. 4b). A new sliding clamp is then positioned at the primer by the clamp-loading complex of DNA polymerase III (Fig. 4c). When synthesis of an Okazaki fragment has been completed, replication halts, and the core subunits of DNA polymerase III dissociate from their β sliding clamp (and from the completed Okazaki fragment) and associate with the new clamp (Fig. 4d, e). This initiates synthesis of a new Okazaki fragment. As noted earlier, the entire complex responsible for coordinated DNA synthesis at a replication fork is a replisome. The proteins acting at the replication fork are summarized in Table 25–4. The replisome promotes rapid DNA synthesis, adding ~1,000 nucleotides/s to each strand (leading and lagging). Once an Okazaki fragment has been completed, its RNA primer is removed and replaced with DNA-by-DNA polymerase I, and the remaining nick is sealed by DNA ligase (Fig. 5).
DNA ligase catalyzes the formation of a phosphodiester bond between a 3 hydroxyl at the end of one DNA strand and a 5 phosphate at the end of another strand. The phosphate must be activated by adenylylation. DNA ligases isolated from viruses and eukaryotes use ATP for this purpose. DNA ligases from bacteria are unusual in that they generally use NAD—a cofactor that normally functions in hydride transfer reactions (see Fig. 5)—as the source of the AMP activating group (Fig. 6). DNA ligase is another enzyme of DNA metabolism that has become an important reagent in recombinant DNA experiments (see Fig. 9–1).
Eventually, the two replication forks of the circular E. coli chromosome meet at a terminus region containing multiple copies of a 20 bp sequence called Ter (for terminus) . The Ter sequences are arranged on the chromosome to create a sort of trap that a replication fork can enter but cannot leave. The Ter sequences function as binding sites for a protein called Tus (terminus utilization substance). The Tus-Ter complex can arrest a replication fork from only one di rection. Only one Tus-Ter complex functions per replication cycle—the complex first encountered by either replication fork. Given that opposing replication forks generally halt when they collide, Ter sequences do not seem essential, but they may prevent overreplication by one replication fork in the event that the other is delayed or halted by an encounter with DNA damage or some other obstacle. So, when either replication fork encounters a functional Tus-Ter complex, it halts; the other fork halts when it meets the first (arrested) fork. The final few hundred base pairs of DNA between these large protein complexes are then replicated (by an as yet unknown mechanism), completing two topologically interlinked (catenated) circular chromosomes . DNA circles linked in this way are known as catenanes. Separation of the catenated circles in E. coli requires topoi somerase IV (a type II topoisomerase). The separated chromosomes then segregate into daughter cells at cell division. The terminal phase of replication of other circular chromosomes, including many of the DNA viruses that infect eukaryotic cells, is similar.
FIGURE 25–5 Final steps in the synthesis of lagging strand segments. RNA primers in the lagging strand are removed by the 5n3 exonuclease activity of DNA polymerase I and replaced with DNA by the same enzyme. The remaining nick is sealed by DNA ligase. The role of ATP or NAD is shown in Figure 6.
FIGURE 25–6 Mechanism of the DNA ligase reaction. In each of the three steps, one phosphodiester bond is formed at the expense of another. Steps 1 and 2 lead to activation of the 5 phosphate in the nick. An AMP group is transferred first to a Lys residue on the en zyme and then to the 5 phosphate in the nick. In step 3, the 3-hydroxyl group attacks this phosphate and displaces AMP, producing a phosphodiester bond to seal the nick. In the E. coli DNA ligase reaction, AMP is derived from NAD+. The DNA ligases isolated from a number of viral and eukaryotic sources use ATP rather than NAD+, and they release pyrophosphate rather than nicotinamide mononucleotide (NMN) in step 1.
