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They were akey to establishing the three-dimensional structure of DNA andyielded clues to how genetic information is encoded in DNA and passedfrom one generation to the next.SeparationbycentrifugationNotradioactiveIRadioactive334DNA Is a Double HelixFigure 12-14 The x-ray diffraction pattern ofDNA. The spots forming a cross in the center denote a helical structure. The heavy bands at thetop and bottom correspond to the recurring bases.Figure 12-15 The Watson-Crick model for thestructure of DNA. The original model proposed thatthere are 10 base pairs or 3.4 nm per turn of thehelix. Subsequent measurements have shown thatthere are 10.5 base pairs or 3.6 nm per turn,(a) Schematic representation, showing dimensionsof the helix, (b) Line model showing the backboneand stacking of the bases, (c) Space-filling model.To shed more light on the structure of DNA, Rosalind Franklin aflMaurice Wilkins used the powerful method of x-ray diffraction (see Be7-3) to analyze DNA crystals.
They showed in the early 1950s thgDNA produces a characteristic x-ray diffraction pattern (Fig. 12-14From this pattern it was deduced that DNA polymers are helical wittwo periodicities along their long axis, a primary one of 0.34 nm andsecondary one of 3.4 nm. The pattern also indicated that the molecidlcontains two strands, a clue that was crucial to determining the strudture.
The problem then was to formulate a three-dimensional model (the DNA molecule that could account not only for the x-ray diffractiadata but also for the specific A = T and G = C base equivalences didcovered by Chargaff and for the other chemical properties of DNA^In 1953 Watson and Crick postulated a three-dimensional modelDNA structure that accounted for all of the available data (Fig.
12-15,It consists of two helical DNA chains coiled around the same axis ttiform a right-handed double helix (see Box 7-1 for an explanation of theright- or left-handed sense of a helical structure). The hydrophilicbackbones of alternating deoxyribose and negatively charged phosphate groups are on the outside of the double helix, facing the sur*rounding water. The purine and pyrimidine bases of both strands arestacked inside the double helix, with their hydrophobic and nearly pla*nar ring structures very close together and perpendicular to the longaxis of the helix. The spatial relationship between these strands creates a major groove and minor groove between the two strands.1Each base of one strand is paired in the same plane with a base of theother strand. Watson and Crick found that the hydrogen-bonded basepairs illustrated in Figure 12-11 are those that fit best within thestructure, providing a rationale for Chargaff s rules.
It is important to:note that three hydrogen bonds can form between G and C, symbolized'G=C, but only two can form between A and T, symbolized A=T. Otherpairings of bases tend (to varying degrees) to destabilize the doublehelical structure.MinorgrooveMajorgroove3.6 nmft'(b)Figure 12-16 Schematic drawing of complementary antiparallel strands of DNA following the pairing rules proposed by Watson and Crick. The basepaired antiparallel strands differ in base composition: the left strand has the composition A3 T2 G2C3; the right, A2 T3 G3 Cv They also differ in sequence when each chain is read in the 5' —> 3' direction. Note the base equivalences: A = T and G =C. In this and following illustrations, the hydrogenbonds between base pairs are often represented bysets of blue lines.In the Watson-Crick structure, the two chains or strands of thehelix are antiparallel; their 5',3'-phosphodiester bonds run in opposite directions.
Later work with DNA polymerases (Chapter 24) provided experimental evidence, confirmed by x-ray crystallography, thatthe strands are indeed antiparallel.To account for the periodicities observed in the x-ray diffractionpattern, Watson and Crick used molecular models to show that thevertically stacked bases inside the double helix would be 0.34 nm apartand that the secondary repeat distance of about 3.4 nm could be accounted for by the presence of 10 (now 10.5) nucleotide residues in eachcomplete turn of the double helix (Fig.
12-15a). As can be seen in Figure 12-16, the two antiparallel polynucleotide chains of double-helicalDNA are not identical in either base sequence or composition. Insteadthey are complementary to each other. Wherever adenine appears inone chain, thymine is found in the other; similarly, wherever guanineis found in one chain, cytosine is found in the other.The DNA double helix or duplex is held together by two sets offorces, as described earlier: hydrogen bonding between complementarybase pairs (Fig. 12-11) and base-stacking interactions.
The specificitythat maintains a given base sequence in each DNA strand is contributed entirely by the hydrogen bonding between base pairs. The basestacking interactions, which are largely nonspecific with respect to theidentity of the stacked bases, make the major contribution to the stability of the double helix.The important features of the double-helical model of DNA structure are supported by much chemical and biological evidence. Moreover, the model immediately suggested a mechanism for the transmission of genetic information.
The essential feature of the model is thecomplementarity of the two DNA strands. Making a copy of this structure (replication) could logically proceed by (1) separating the twostrands and (2) synthesizing a complementary strand for each by joining nucleotides in a sequence specified by the base-paring rules statedabove. Each preexisting strand could function as a template to guidethe synthesis of the complementary strand (Fig. 12-17). These expectations have been experimentally confirmed, and this discovery was arevolution in our understanding of DNA metabolism.Figure 12-17 Replication of DNA as suggested byWatson and Crick. The parent strands become separated, and each forms the template for biosynthesis of a complementary daughter strand (in red).5'5'ParentstrandDaughterstrandsParentstrand335Part II Structure and Catalysis336DNA Can Occur in Different Structural Forms2.3 nmA formB formZ formFigure 12-18 Comparison of the A, B, and Z formsof DNA.
There are 24 base pairs in each of thestructures shown.DNA is a remarkably flexible molecule. Considerable rotation is possible around a number of bonds in the sugar-phosphate backbone, andthermal fluctuation can produce bending, stretching, and unpairing(melting) in the structure. Many significant deviations from theWatson-Crick DNA structure are found in cellular DNA, and some orall of these may play important roles in DNA metabolism. These structural variations generally do not affect the key properties of DNA defined by Watson and Crick: strand complementarity, antiparallelstrands, and the requirement for A=T and G=C base pairs.The Watson-Crick structure is also referred to as B-form DNA.The B form is the most stable structure for a random-sequence DNAmolecule under physiological conditions, and is therefore the standardpoint of reference in any study of the properties of DNA.
Two DNAstructural variants that have been well characterized in crystal structures are the A and Z forms (Fig. 12-18). The A form is favored in manysolutions that are relatively devoid of water. The DNA is still arrangedin a right-handed double helix, but the rise per base pair is 0.23 nmand the number of base pairs per helical turn is 11, relative to the0.34 nm rise and 10.5 base pairs per turn found in B-DNA. For a givenDNA molecule, the A form will be shorter and have a greater diameterthan the B form. The reagents used to promote crystallization of DNAtend to dehydrate it, and this leads to a tendency for many DNAs tocrystallize in the A form.Z-form DNA is a more radical departure from the B structure; themost obvious distinction is the left-handed helical rotation.
There are12 base pairs per helical turn, with a rise of 0.38 nm per base pair. TheDNA backbone takes on a zig-zag appearance. Certain nucleotide sequences fold up into left-handed Z helices more readily than do others.Prominent examples are sequences in which pyrimidines alternatewith purines, especially alternating C and G or 5-methyl-C and G.Whether A-form DNA actually occurs in cells is uncertain, but there isevidence for some short stretches (tracts) of Z-DNA in both prokaryotesand eukaryotes.
These Z-DNA tracts may play an as yet undefined rolein the regulation of the expression of some genes or in genetic recombination.Certain DNA Sequences Adopt Unusual StructuresA number of other sequence-dependent structural variations havebeen detected that may serve locally important functions in DNA metabolism. For example, some sequences cause bends in the DNA helix.Bends are produced whenever four or more adenine residues appearsequentially in one of the two strands (Fig. 12-19). Six adenines in arow produce a bend of about 18°. The bending observed with this andother sequences may be important in the binding of some proteins toDNA.Figure 12-19 A model for the bending of DNAproduced by poly(A) tracts.
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