DNA Replication:- DNA Replication Follows a Set of Fundamental Rules
Early research on bacterial DNA replication and its en zymes helped to establish several basic properties that have proven applicable to DNA synthesis in every organism.
DNA Replication Is Semiconservative Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules, each with one new strand and one old strand. This is semiconservative replication. Watson and Crick proposed the hypothesis of semi conservative replication soon after publication of their 1953 paper on the structure of DNA, and the hypothesis was proved by ingeniously designed experiments carried out by Matthew Meselson and Franklin Stahl in 1957. Meselson and Stahl grew E. coli cells for many generations in a medium in which the sole nitrogen source (NH4Cl) contained 15N, the “heavy” isotope of nitrogen, instead of the normal, more abundant “light” isotope, 14N. The DNA isolated from these cells had a density about 1% greater than that of normal [14N] DNA (Fig. 1a). Although this is only a small difference, a mixture of heavy [15N] DNA and light [14N] DNA can be separated by centrifugation to equilibrium in a cesium chloride density gradient.
The E. coli cells grown in the 15N medium were transferred to a fresh medium containing only the 14N isotope, where they were allowed to grow until the cell population had just doubled. The DNA isolated from these first-generation cells formed a single band in the CsCl gradient at a position indicating that the double helical DNA molecules of the daughter cells were hybrids containing one new 14N strand and one parent 15N strand (Fig. 1b).
This result argued against conservative replication, an alternative hypothesis in which one progeny DNA molecule would consist of two newly synthesized DNA strands and the other would contain the two parent strands; this would not yield hybrid DNA molecules in the Meselson-Stahl experiment. The semiconservative replication hypothesis was further supported in the next step of the experiment (Fig. 1c). Cells were again allowed to double in number in the 14N medium. The isolated DNA product of this second cycle of replication exhibited two bands in the density gradient, one with a density equal to that of light DNA and the other with the density of the hybrid DNA observed after the first cell doubling.
Replication Begins at an Origin and Usually Proceeds Bidirectionally Following the confirmation of a semiconservative mechanism of replication, a host of questions arose. Are the parent DNA strands completely unwound before each is replicated? Does replication begin at random places or at a unique point? After initiation at any point in the DNA, does replication proceed in one di rection or both? An early indication that replication is a highly coordinated process in which the parent strands are simultaneously unwound and replicated was provided by John Cairns, using autoradiography. He made E. coli DNA radioactive by growing cells in a medium containing thymidine labeled with tritium (3H). When the DNA was carefully isolated, spread, and overlaid with a photographic emulsion for several weeks, the radioactive thymidine residues generated “tracks” of silver grains in the emulsion, producing an image of the DNA molecule. These tracks revealed that the intact chromosome of E. coli is a single huge circle, 1.7 mm long. Radioactive DNA isolated from cells during replication showed an extra loop (Fig. 2a). Cairns concluded that the loop resulted from the formation of two radioactive daughter strands, each complementary to a parent strand. One or both ends of the loop are dynamic points, termed replication forks, where parent DNA is being un wound and the separated strands quickly replicated. Cairns’s results demonstrated that both DNA strands are replicated simultaneously, and a variation on his experiment (Fig.2b) indicated that replication of bacterial chromosomes is bidirectional: both ends of the loop have active replication forks. The determination of whether the replication loops originate at a unique point in the DNA required land marks along the DNA molecule. These were provided by a technique called denaturation mapping, developed by Ross Inman and colleagues. Using the 48,502 bp chromosome of bacteriophage , Inman showed that DNA could be selectively denatured at sequences un usually rich in A=T base pairs, generating a reproducible pattern of single-strand bubbles . Isolated DNA containing replication loops can be partially denatured in the same way. This allows the position and progress of the replication forks to be measured and mapped, using the denatured regions as points of reference. The technique revealed that in this system the replication loops always initiate at a unique point, which was termed an origin. It also confirmed the earlier observation that replication is usually bidirectional. For circular DNA molecules, the two replication forks meet at a point on the side of the circle opposite to the origin. Specific origins of replication have since been identified and characterized in bacteria and lower eukaryotes.
DNA Synthesis Proceeds in a 5→3 Direction and Is Semidis continuous A new strand of DNA is always synthesized in the 5n3 direction, with the free 3 OH as the point at which the DNA is elongated . Because the two DNA strands are antiparallel, the strand serving as the template is read from its 3 end toward its 5 end. If synthesis always proceeds in the 5→3 direction, how can both strands be synthesized simultaneously? If both strands were synthesized continuously while the replication fork moved, one strand would have to undergo 3n5 synthesis. This problem was resolved by Reiji Okazaki and colleagues in the 1960s. Okazaki found that one of the new DNA strands is synthesized in short pieces, now called Okazaki fragments. This work ultimately led to the conclusion that one strand is synthesized continuously and the other discontinuously (Fig. 3). The continuous strand, or leading strand, is the one in which 5→3 synthesis proceeds in the same direction as replication fork movement. The dis continuous strand, or lagging strand, is the one in which 5n3 synthesis proceeds in the direction oppo site to the direction of fork movement. Okazaki fragments range in length from a few hundred to a few thou sand nucleotides, depending on the cell type. As we shall see later, leading and lagging strand syntheses are tightly coordinated.
FIGURE 1 The Meselson-Stahl experiment. (a) Cells were grown for many generations in a medium containing only heavy nitrogen, 15N, so that all the nitrogen in their DNA was 15N, as shown by a single band (blue) when centrifuged in a CsCl density gradient. (b) Once the cells had been transferred to a medium containing only light nitrogen, 14N, cellular DNA isolated after one generation equilibrated at a higher position in the density gradient (purple band). (c) Continuation of replication for a second generation yielded two hybrid DNAs and two light DNAs (red), confirming semiconservative replication.
FIGURE 2 Visualization of bidirectional DNA replication. Replication of a circular chromosome produces a structure resembling the Greek letter theta (Ө). (a) Labeling with tritium (3H) shows that both strands are replicated at the same time (new strands shown in red). The electron micrographs illustrate the replication of a circular E. coli plasmid as visualized by autoradiography. (b) Addition of 3H for a short period just before the reaction is stopped allows a distinction to be made between unidirectional and bidirectional replication, by determining whether label (red) is found at one or both replication forks in autoradiograms. This technique has revealed bidirectional replication in E. coli, Bacillus subtilis, and other bacteria.
FIGURE 3 Defining DNA strands at the replication fork. A new DNA strand (red) is always synthesized in the 5→3 direction. The template is read in the opposite direction, 3→5. The leading strand is continuously synthesized in the direction taken by the replication fork. The other strand, the lagging strand, is synthesized discontinuously in short pieces (Okazaki fragments) in a direction opposite to that in which the replication fork moves. The Okazaki fragments are spliced together by DNA ligase. In bacteria, Okazaki fragments are ~1,000 to 2,000 nucleotides long. In eukaryotic cells, they are 150 to 200 nucleotides long.
