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Cells were grownfor many generations in a medium containing onlyheavy nitrogen, 15N, so that all the nitrogen in theDNA was 15N. The cells were then transferred to amedium containing only light nitrogen, 14N, and thedensity of the DNA was monitored closely for thenext two cell generations. Cellular DNA was isolated after the first and second generations andcentrifuged to equilibrium in a CsCl density gradient. The [15N]DNA (shown in blue) came to equilibrium at a lower position in the CsCl gradient than[14N]DNA (shown in red).
Hybrid DNA equilibratedin an intermediate position. If DNA replicationwere conservative, each of the two heavy strands ofparental DNA would be replicated to yield the original heavy duplex DNA and a DNA duplex containing two new light strands. Continuation of conservative replication would yield in the next generationone heavy DNA and three light DNAs but nohybrid DNAs.
The Meselson-Stahl experiment,however, showed that replication is semiconservative, resulting in two daughter duplexes each containing one parental heavy strand and one new lightstrand. The next generation yielded two hybridDNAs and two light DNAs.818Part IV Information PathwaysFigure 24-3 Replication of a circular chromosomeproduces a structure resembling the Greek lettertheta (0). (a) Labeling with tritium (3H) shows thatboth strands are replicated at the same time (newstrands shown in red).
The electron micrographs illustrate the replication of a circular E. coli plasmidas visualized by autoradiography. (b) Addition of3H for a short period just before the reaction isstopped allows a distinction to be made betweenunidirectional and bidirectional replication, by determining whether label (in red) is found at one orboth replication forks seen in autoradiograms. Thistechnique has revealed bidirectional replication inE. coli, B.
subtihs, and other bacteria, (c) Autoradiogram of a replicating E. coli chromosome takenfrom a culture grown for two generations in[3H]thymidine.Replication Begins at an Origin and Usually Proceeds Bidirectionally A host of questions now arises. Are the parental DNA strandscompletely unwound before each is replicated? Does replication beginat random places or at a unique point? After initiation at any point inthe DNA, does replication proceed in one direction or both? An earlyindication that replication is a highly coordinated process in which theparental strands are unwound and replicated simultaneously was provided by John Cairns using the technique of autoradiography.
Hemade the DNA of E. coli cells radioactive by growing them in a mediumcontaining thymidine labeled with tritium (3H). When the DNA wascarefully isolated, spread, and overlaid with a photographic emulsion,and left for several weeks, the radioactive thymidine residues generated "tracks" of silver grains in the emulsion, producing an image ofthe DNA molecule. These tracks revealed that the intact chromosomeof E. coli is a single giant circle, 1.7 mm long (see Fig. 23-2). Radioactive DNA isolated from cells during replication showed an extra radioactive loop (Fig.
24-3). The amount of radioactivity in the loop relativeto the remainder of the DNA led Cairns to conclude that the loop in theDNA was the result of the formation of two radioactive daughterstrands, each complementary to a parent strand. One or both ends ofthe loop are dynamic points, termed replication forks, where parental DNA is being unwound and the separated strands quickly repli-\r " .
,\ *•Origin[UnidirectionalI%Origin(b)(c)Chapter 24 DNA Metabolism819cated. This demonstrated that both DNA strands are replicated simultaneously, and a variation of this experiment (Fig. 24-3b) indicatedthat replication of bacterial chromosomes is bidirectional: both ends ofthe loop have active replication forks.To determine whether the loops originated at a unique point in theDNA, landmarks were needed in the DNA "string." These were provided by a technique called denaturation mapping, developed byRoss Inman and colleagues.
Using the 48,502 base pair chromosomefrom bacteriophage A, Inman showed that DNA could be selectivelydenatured at sequences unusually rich in A=T base pairs. This generates a reproducible pattern of single-stranded bubbles (see Fig. 12-30).When isolated DNAs containing replication loops are partially denatured in this way, the progress of the replication forks can be measuredand mapped using the denatured regions as points of reference. Thetechnique revealed that the replication loops always initiate at aunique point, called an origin. In addition, this work reinforced theearlier observation that replication is usually bidirectional. For circular DNA molecules, the two replication forks meet at a point on theside of the circle opposite to the origin.DNA Synthesis Proceeds in a 5'-*3f Direction and Is Semidiscontinuous A new strand of DNA is always synthesized in the 5'—>3' direction (the 5' and 3' ends of a DNA strand are defined as shown in Figure12-7).
Because the two DNA strands are antiparallel, the strand acting as template is being read from its 3' end toward its 5' end.If synthesis always proceeds in the 5'—»3' direction, how can bothstrands be synthesized simultaneously? If both were synthesized continuously as the replication fork moved, one would have to undergo3'—»5' synthesis.
This problem was resolved by Reiji Okazaki and colleagues in the 1960s. Okazaki found that one of the new DNA strandsis synthesized in short pieces, now called Okazaki fragments. Thiswork ultimately led to the conclusion that one strand is synthesizedcontinuously and the other discontinuously (Fig. 24-4). The continuous or leading strand is the one in which 5'^>3' synthesis proceeds inthe same direction as replication fork movement. The discontinuous orlagging strand is the one in which 5'—»3' synthesis proceeds in thedirection opposite to the direction of fork movement.
Okazaki fragments range in length from a few hundred to a few thousand nucleotides, depending on the cell type.Leadingstrand5'3'LaggingstrandFigure 24-4 A new DNA strand (red) is alwayssynthesized in the 5'—>3' direction. The template iscopied in the opposite direction: 3'-»5'. The strandthat is continuously synthesized (in the directiontaken by the replication fork) is the leading strand.The other strand, the lagging strand, is synthesizeddiscontinuously in short pieces (Okazaki fragments)in a direction opposite to the direction of replicationfork movement.
The Okazaki fragments are thenspliced together by DNA ligase. In bacteria theOkazaki fragments are about 1,000 to 2,000 nucleotides long. In eukaryotic cells they are 150 to 200nucleotides long.820Part IV Information PathwaysDNA Is Synthesized by DNA PolymerasesThe search for an enzyme that could synthesize DNA was initiated in1955 by Arthur Kornberg and colleagues. This work led to the purification and characterization of DNA polymerase from E. coli cells, asingle-polypeptide enzyme now called DNA polymerase I (Mr103,000).
Much later, it was found that E. coli contains at least twoother distinct DNA polymerases, which will be described below.Detailed studies of DNA polymerase I revealed features of theDNA synthetic process that have proven to be common to all DNApolymerases. The fundamental reaction is a nucleophilic attack by the3'-hydroxyl group of the nucleotide at the 3' end of the growing strandon the 5'-a-phosphorus of the incoming deoxynucleoside 5'-triphosphate (Fig.
24-5). Inorganic pyrophosphate is released in the reaction.The general reaction equation is(dNMP)n + dNTPDNAArthur Kornberg(24-1)n+1LengthenedDNAwhere dNMP and dNTP are deoxynucleoside 5'-monophosphate and5'-triphosphate, respectively.GrowingDNAstrand(primer)5' yFigure 24-5 Elongation of a DNA chain. A singleunpaired strand is required to act as template, anda primer strand is needed to provide a free 3' endto which new nucleotide units are added.
Each incoming nucleotide is selected by virtue of base pairing to the appropriate nucleotide in the templatestrand.OTemplateDNAstrandN3'O"O—P—O—P—O—P —QO-O"Incomingdeoxynucleoside5'-triphosphateEarly work on DNA polymerase I led to the definition of two central requirements for DNA polymerization. First, all DNA polymerasesrequire a template (Fig. 24-5). The polymerization reaction is guidedby a template DNA strand according to the base-pairing rules predicted by Watson and Crick: where a guanine is present in the template, a cytosine is added to the new strand, and so on.
This was aparticularly important discovery, not only because it provided a chemical basis for semiconservative DNA replication, but because it represented the first example of the use of a template to guide a biosyntheticreaction. Second, a primer is required. A primer is a segment of newstrand (complementary to the template) with a 3'-hydroxyl group towhich nucleotides can be added. The 3' end of the primer is called theprimer terminus.
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