DNA Structure and Replication PDF

DNA STRUCTURE AND REPLICATION  Overview of DNA replication As DNA pol III moves forward, the double helix is continuously unwinding ahead of the enzyme to expose further lengths of single DNA strands that will act as templates (Figure 17-16). DNA pol III acts at the replica...

DNA STRUCTURE AND REPLICATION  Overview of DNA replication As DNA pol III moves forward, the double helix is continuously unwinding ahead of the enzyme to expose further lengths of single DNA strands that will act as templates (Figure 17-16). DNA pol III acts at the replication fork, the zone where the double helix is unwinding. However, because DNA polymerase always adds nucleotides at the 3 growing tip, only one of the two antiparallel strands can serve as a template for replication in the direction of the replication fork. For this strand, synthesis can take place in a smooth continuous manner in the direction of the fork; the new strand synthesized on this template is called the leading strand. Synthesis on the other template also takes place at 3 growing tips, but this synthesis is in the “wrong” direction, because, for this strand, the 5-to-3 direction of synthesis is away from the replication fork (see Figure 7-16). As we will see, the nature of the replication machinery requires that synthesis of both strands take place in the region of the replication fork. Therefore, synthesis moving away from the growing fork cannot go on for long. It must be in short segments: polymerase synthesizes a segment, then moves back to the segment’s 5 end, where the growing fork has exposed new template, and begins the process again. These short (1000–2000 nucleotides) stretches of newly synthesized DNA are called Okazaki fragments. Overview of DNA replication Another problem in DNA replication arises because DNA polymerase can extend a chain but cannot start a chain. Therefore, synthesis of both the leading strand and each Okazaki fragment must be initiated by a primer, or short chain of nucleotides, that binds with the template strand to form a segment of duplex DNA. The primer inDNA replication can be seen in Figure 7-17. The primers are synthesized by a set of proteins called a primosome, of which a central component is an enzyme called primase, a type of RNA polymerase. Primase synthesizes a short (8–12 nucleotides) stretch of RNA complementary to a specific region of the chromosome. On the leading strand, only one initial primer is needed because, after the initial priming, the growing DNA strand serves as the primer for continuous addition. However, on the lagging strand, every Okazaki fragment needs its own primer. The RNA chain composing the primer is then extended as a DNA chain by DNA pol III.  Overview of DNA replication A different DNA polymerase, pol I, removes the RNA primers and fills in the resulting gaps with DNA. As mentioned earlier, pol I is the enzyme originally purified by Kornberg. Another enzyme, DNA ligase, joins the 3 end of the gap-filling DNA to the 5 end of the downstream Okazaki fragment. The new strand thus formed is called the lagging strand. DNA ligase joins broken pieces of DNA by catalyzing the formation of a phosphodiester bond between the 5-phosphate end of one fragment and the adjacent 3-OH group of another fragment. One of the hallmarks of DNA replication is its accuracy, also called fidelity: overall, there is less than one error per 1010 nucleotides inserted. Part of the reason for the accuracy of DNA replication is that both DNA pol I and DNA pol III possess 3-to-5 exonuclease activity, which serves a “proofreading” function by excising mismatched bases that were inserted erroneously. Strains lacking a functional 3-to-5 exonuclease have a higher rate of mutation. In addition, because primase lacks a proofreading function, the RNA primer is more likely than DNA to contain errors. To maintain the high fidelity of replication, it is essential that the RNA primers at the ends of Okazaki fragments be removed and replaced with DNA by DNA pol I.  The replisome: A remarkable replication machine The second hallmark of DNA replication is speed. The time needed for E. coli to replicate its chromosome can be as short as 20 minutes. Therefore, its genome of about 5 million base pairs must be copied at a rate of more than 2000 nucleotides per second. From the experiment of Cairns, we know that E. coli uses only two replication forks to copy its entire genome. Thus, each fork must be able to move at a rate of as many as 1000 nucleotides per second. What is remarkable about the entire process of DNA replication is that it does not sacrifice speed for accuracy. How can it maintain both speed and accuracy, given the complexity of the reactions that need to be carried out at the replication fork? The answer is that DNA polymerase is actually part of a large “nucleoprotein” complex that coordinates the activities at the replication fork. This complex, called the replisome, is an example of a “molecular machine”. You will encounter other examples in later chapters. The discovery that most of the major functions of cells—replication, transcription, and translation, for example—are carried out by large multisubunit complexes has changed the way that we think about the cell.  The replisome: A remarkable replication machine To begin to understand why, let’s look at the replisome more closely. Some of the interacting components of the replisome in E. coli are shown in Figure 7-18. At the replication fork, the catalytic core of DNA pol III is actually part of a much larger complex, called the pol III holoenzyme, which consists of two catalytic cores and many accessory proteins. One of the catalytic cores handles the synthesis of the leading strand while the other handles lagging strand synthesis. Some of the accessory proteins (not visible in Figure 7-18) form a connection that bridges the two catalytic cores, thus coordinating the synthesis of the leading and lagging strands. The lagging strand is shown looping around so that the replisome can coordinate the synthesis of both strands and move in the direction of the replication fork. Also shown is an important accessory protein called the sliding clamp, which encircles the DNA like a donut. Its association with the clamp protein keeps pol III attached to the DNA molecule. Thus, pol III is transformed from an enzyme that can add only 10 nucleotides before falling off the template (termed a distributive enzyme) to an enzyme that stays at the moving fork and adds tens of thousands of nucleotides (a processive enzyme). In sum, through the action of accessory proteins, synthesis of both the leading and the lagging strands is rapid and highly coordinated. Note also that primase, the enzyme that synthesizes the RNA primer, is not touching the clamp protein. Therefore, primase will act as a distributive enzyme it adds only a few ribonucleotides before dissociating from the template. This mode of action makes sense because the primer need be only long enough to form a suitable duplex starting point for DNA pol III.  Unwinding the double helix When the double helix was proposed in 1953, a major objection was that the replication of such a structure would require the unwinding of the double helix at the replication fork and the breaking of the hydrogen bonds that hold the strands together. How could DNA be unwound so rapidly and, even if it could, wouldn’t that overwind the DNA behind the fork and make it hopelessly tangled? We now know that the replisome contains two classes of proteins that open the helix and prevent overwinding: they are helicases and topoisomerases, respectively. Helicases are enzymes that disrupt the hydrogen bonds that hold the two strands of the double helix together. Like the clamp protein, the helicase fits like a donut around the DNA; from this position, it rapidly unzips the double helix ahead of DNA synthesis. The unwound DNA is stabilized by single-strand-binding (SSB) proteins, which bind to single-stranded DNA and prevent the duplex from reforming. Circular DNA can be twisted and coiled, much like the extra coils that can be introduced into arubber band. Untwisting of the replication fork by helicases causes extra twisting at other regions, and coils called supercoils form to release the strain of the extra twisting. Both the twists and the supercoils must be removed to allow replication to continue. This supercoiling can be created or relaxed by enzymes termed topoisomerases, an example of which is DNA gyrase (Figure 7-19). Topoisomerases relax supercoiled DNA by breaking either a single DNA strand or both strands, which allows DNA to rotate into a relaxed molecule. Topoisomerase finishes by rejoining the strands of the now relaxed DNA molecule. The eukaryotic replisome DNA replication in both prokaryotes and eukaryotes uses a semiconservative mechanism and employs leading- and lagging-strand synthesis. For this reason, it should not come as a surprise that the components of the replisome in prokaryotes and eukaryotes are very similar. However, as organisms increase in complexity, the number of replisome components also increases. There are now known to be 13 components of the E. coli replisome and at least 27 in the replisomes of yeast and mammals. One reason for the added complexity of the eukaryotic replisome is the higher complexity of the eukaryotic template. Recall that, unlike the bacterial chromosome, eukaryotic chromosomes exist in the nucleus as chromatin. As described in Chapter 3, the basic unit of chromatin is the nucleosome, which consists of DNA wrapped around histone proteins. Thus, the replisome has to not only copy the parental strands, but also disassemble the nucleosomes in the parental strands and reassemble them in the daughter molecules. This is done by randomly distributing the old histones (from the existing nucleosomes) to daughter molecules and delivering new histones in association with a protein called chromatin assembly factor 1 (CAF-1) to the replisome. CAF- 1 binds to histones and targets them to the replication fork, where they can be assembled together with newly synthesized DNA. CAF-1 and its cargo of histones arrive at the replication fork by binding to the eukaryotic version of the clamp protein, called proliferating cell nuclear antigen (PCNA) (Figure 7-20).  Assembling the replisome: replication initiation Assembly of the replisome in both prokaryotes and eukaryotes is an orderly process that begins at precise sites on the chromosome (called origins) and takes place only at certain times in the life of the cell. Prokaryotic origins of replication E. coli replication begins from a fixed origin (called oriC) and then proceeds in both directions (with moving forks at both ends, as previously shown in Figure 7-14) until the forks merge. Figure 7-21a shows the process. The first step in the assembly of the replisome is the binding of a protein called DnaA to a specific 13-bp sequence (called a “DnaA box”) that is repeated five times in oriC. In response to the binding of DnaA, the origin is unwound at a cluster of A and T nucleotides. Recall that AT base pairs are held together only with two hydrogen bonds, whereas GC base pairs are held together by three. Thus, it is easier to separate (melt) the double helix at stretches of DNA that are enriched in A and T bases. After unwinding begins, additional DnaA proteins bind to the newly unwound single-stranded regions. With DnaA coating the origin, two helicases (the DnaB protein) now bind and slide in a 5-to-3 direction to begin unzipping the helix at the replication fork. Primase and DNA pol III holoenzyme are now recruited to the replication fork by protein–protein interactions and DNA synthesis begins. You may be wondering why DnaA is not present in Figure 7-18 (the replisome machine). The answer is that, although it is necessary for the assembly of the replisome, it is not part of the replication machinery. Rather, its job is to bring the replisome to the correct place in the circular chromosome for the initiation of replication.  Eukaryotic origins of replication Bacteria such as E. coli usually complete a replication division cycle in from 20 to 40 minutes but, in eukaryotes, the cycle can vary from 1.4 hours in yeast to 24 hours in cultured animal cells and may last from 100 to 200 hours in some cells. Eukaryotes have to solve the problem of coordinating the replication of more than one chromosome, as well as the problem of replicating the complex structure of the chromosome itself. The origins of the simple eukaryote, yeast, are very much like oriC in E. coli. They have AT-rich regions that melt when an initiator protein binds to adjacent binding sites. Origins of replication are not well characterized in higher organisms, but they are known to be much longer, possibly as long as thousands or tens of thousands of nucleotides. Unlike prokaryotic chromosomes, each chromosome has many replication origins in order to replicate the much larger eukaryotic genomes quickly. Approximately 400 replication origins are dispersed throughout the 16 chromosomes of yeast, and there are estimated to be thousands of growing forks in the 23 chromosomes of humans. Thus, in eukaryotes, replication proceeds in both directions from multiple points of origin (Figure 7-22). The double helices that are being produced at each origin of replication elongate and eventually join one another. When replication of the two strands is complete, two identical daughter molecules of DNA result.