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JWCL281_c28_1107-1142.qxd 4/22/10 9:17 AM Page 1141

1142 Chapter 28. Nucleotide Metabolism

1. Azaserine (O-diazoacetyl-L-serine) and 6-diazo-5-oxo-L-

norleucine (DON)

are glutamine analogs. They form covalent bonds to nucleophiles

at the active sites of enzymes that bind glutamine, thereby irre-

versibly inactivating these enzymes. Identify the nucleotide

biosynthesis intermediates that accumulate in the presence of ei-

ther of these glutamine antagonists.

2. Suggest a mechanism for the AIR synthetase reaction

(Fig. 28-2, Reaction 6).

*3. What is the energetic price, in ATPs, of synthesizing the hy-

poxanthine residue of IMP from CO

and NH

⫹

4. Why is deoxyadenosine toxic to mammalian cells?

5. Indicate which of the following substances are mechanism-

based inhibitors and explain your reasoning. (a) Tosyl-

L-phenyl-

alanine chloromethylketone with chymotrypsin (Section 15-3Ab).

(b) Trimethoprim with bacterial dihydrofolate reductase. (c) The

␦-lactone analog of (NAG)

with lysozyme (Section 15-2Cb). (d)

Allopurinol with xanthine oxidase.

6. Why do individuals who are undergoing chemotherapy

with cytotoxic (cell killing) agents such as FdUMP or methotrex-

ate temporarily go bald?

6-Diazo-5-oxo-L-norleucine (DON)

Azaserine

NNCH

COO

7. Normal cells die in a nutrient medium containing thymidine

and methotrexate that supports the growth of mutant cells defec-

tive in thymidylate synthase. Explain.

8. FdUMP and methotrexate, when taken together, are less ef-

fective chemotherapeutic agents than when either drug is taken

alone. Explain.

9. Some microorganisms lack DHFR activity, but their

thymidylate synthase has an FAD cofactor.What is the function of

the FAD?

10. Why is gout more prevalent in populations that eat rela-

tively large amounts of meat?

11. Gout resulting from the de novo overproduction of

purines can be distinguished from gout caused by impaired excre-

tion of uric acid by feeding a patient

N-labeled glycine and de-

termining the distribution of

N in his or her excreted uric acid.

What isotopic distributions are expected for each type of defect?

12. 6-Mercaptopurine,

after its conversion to the corresponding nucleotide through sal-

vage reactions, is a potent competitive inhibitor of IMP in the

pathways for AMP and GMP biosynthesis. It is therefore a clini-

cally useful anticancer agent.The chemotherapeutic effectiveness

of 6-mercaptopurine is enhanced when it is administered with al-

lopurinol. Explain the mechanism of this enhancement.

6-Mercaptopurine

PROBLEMS

JWCL281_c28_1107-1142.qxd 7/21/10 7:10 PM Page 1142

PART V

EXPRESSION AND

TRANSMISSION

OF GENETIC

INFORMATION

Schematic diagram of the

eukaryotic preinitiation

complex that is required

for the transcription of

DNA to messenger RNA.

The TATA-box binding

protein is shown in orange.

JWCL281_c29_1143-1172.qxd 7/9/10 12:46 AM Page 1143

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1145

CHAPTER 29

Nucleic Acid

Structures

1 Double Helical Structures

A. B-DNA

B. Other Nucleic Acid Helices

2 Forces Stabilizing Nucleic Acid Structures

A. Sugar–Phosphate Chain Conformations

B. Base Pairing

C. Base Stacking and Hydrophobic Interactions

D. Ionic Interactions

3 Supercoiled DNA

A. Superhelix Topology

B. Measurements of Supercoiling

C. Topoisomerases

The structure and properties of DNA are introduced in

Section 5-3. In this chapter we extend this discussion with

emphasis on DNA; the structures of RNAs are detailed in

Sections 31-4A and 32-2B. Methods of purifying, sequenc-

ing, and chemically synthesizing nucleic acids are discussed

in Sections 6-6, 7-2, and 7-6, and recombinant DNA tech-

niques are discussed in Section 5-5. Bioinformatics, as it

concerns nucleic acids, is outlined in Section 7-4, and the

Nucleic Acid Database is described in Section 8-3Cb.

1 DOUBLE HELICAL STRUCTURES

See Guided Exploration 23: DNA structures Double helical

DNA has three major helical forms, B-DNA,A-DNA, and

Z-DNA, whose structures are depicted in Fig. 29-1. In this

section we discuss the major characteristics of each of these

helical forms as well as those of double helical RNA and

DNA–RNA hybrid helices.

A. B-DNA

The structure of B-DNA (Fig. 29-1, middle panels), the bi-

ologically predominant form of DNA, is described in Sec-

tion 5-3A.To recapitulate (Table 29-1), B-DNA consists of

a right-handed double helix whose two antiparallel

sugar–phosphate chains wrap around the periphery of the

helix. Its aromatic bases (A, T, G, and C), which occupy

the core of the helix, form complementary A ⴢ T and G ⴢ C

Watson–Crick base pairs (Fig. 5-12), whose planes are

nearly perpendicular to the axis of the double helix. Neigh-

boring base pairs, whose aromatic rings are 3.4 Å thick, are

stacked in van der Waals contact, with the helix axis passing

through the middle of each base pair. B-DNA is ⬃20 Å in

diameter and has two deep grooves between its

sugar–phosphate chains: the relatively narrow minor

groove, which exposes that edge of the base pairs from

which the glycosidic bonds (the bonds from the base N to

the ribose C1¿) extend (toward the bottom of Fig. 5-12), and

the relatively wide major groove, which exposes the oppo-

site edge of each base pair (toward the top of Fig. 5-12).

Canonical (ideal) DNA has a helical twist of 10 base pairs

(bp) per turn and hence a pitch (rise per turn) of 34 Å.

The Watson–Crick base pairs in either orientation are

structurally interchangeable, that is, A ⴢ T, T ⴢ A, G ⴢ C, and

1145

There are two classes of nucleic acids, deoxyribonucleic

acid (DNA) and ribonucleic acid (RNA). DNA is the

hereditary molecule in all cellular life-forms, as well as in

many viruses. It has but two functions:

1. To direct its own replication during cell division.

2. To direct the transcription of complementary mole-

cules of RNA.

RNA, in contrast, has more varied biological functions:

1. The RNA transcripts of DNA sequences that specify

polypeptides, messenger RNAs (mRNAs), direct the ribo-

somal synthesis of these polypeptides in a process known

as translation.

2. The RNAs of ribosomes, which are about two-thirds

RNA and one-third protein, have functional as well as

structural roles.

3. During protein synthesis, amino acids are delivered

to the ribosome by molecules of transfer RNA (tRNA).

4. Certain RNAs are associated with specific proteins

to form ribonucleoproteins that participate in the post-

transcriptional processing of other RNAs.

5. A variety of short RNAs participate in the control of

eukaryotic gene expression and in protection against

viruses, a phenomenon known as RNA interference

(RNAi).

6. In many viruses, RNA, not DNA, is the carrier of

hereditary information.

JWCL281_c29_1143-1172.qxd 7/9/10 12:46 AM Page 1145

C ⴢ G can replace each other in the double helix without al-

tering the positions of the sugar–phosphate backbones’ C1¿

atoms. In contrast, any other combination of bases would

significantly distort the double helix since the formation of

a non-Watson–Crick base pair would require considerable

reorientation of the DNA’s sugar–phosphate backbones.

a. Real DNA Deviates from the Ideal

Watson–Crick Structure

The DNA samples that were available when James Wat-

son and Francis Crick formulated the Watson–Crick struc-

ture in 1953 were extracted from cells and hence consisted

of molecules of heterogeneous lengths and base sequences.

1146 Chapter 29. Nucleic Acid Structures

Minor

groove

Major

groove

A-DNA

(a)

Minor

groove

Major

groove

B-DNA

Minor

groove

Z-DNA

Major

groove

(b)

Figure 29-1 Structures of A-, B-, and Z-DNAs. (a) Ball-and

stick drawings viewed perpendicular to the helix axis.The

sugar–phosphate backbones, which wind about the periphery of

each molecule, are outlined by green ribbons, and the bases,

which occupy its core, are red. Note that the two sugar–phosphate

chains in each helix run in opposite directions so as to form right-

handed double helices in A- and B-DNAs and a left-handed

double helix in Z-DNA. (b) Views along the helix axis. The ribose

ring O atoms are red and the nucleotide pair closest to the viewer

is white. Note that the helix axis passes far “above” the major

groove of A-DNA, through the base pairs of B-DNA, and

through the edge of the minor groove of Z-DNA. Consequently,

A-DNA has a hollow core whereas B- and Z-DNAs have solid

cores.Also note that the deoxyribose residues in A- and B-DNAs

have the same conformation in each helix, but those in Z-DNA

have two different conformations so that alternate ribose residues

JWCL281_c29_1143-1172.qxd 8/2/10 8:06 PM Page 1146

-DN

B-DN

-DN

(c)

(d)

Such elongated molecules do not crystallize, but can be

drawn into threadlike fibers in which the helix axes of the

DNA molecules are all approximately parallel to the fiber

axis but are poorly aligned, if at all, in any other way. The

X-ray diffraction patterns of such fibers provide only crude,

low-resolution images in which the base pair electron den-

sity is the average electron density of all the base pairs in

the fiber.The Watson–Crick structure was based, in part, on

the X-ray fiber diffraction pattern of B-DNA (Fig. 5-10).

By the late 1970s, advances in nucleic acid chemistry

permitted the synthesis and crystallization of ever longer

oligonucleotides of defined sequences (Section 7-6A),

Section 29-1. Double Helical Structures 1147

lie at different radii. (c) Space-filling models viewed perpendicular

to the helix axis and colored according to atom type (C white,

N blue, O red, and P orange). (d) Space-filling models viewed

along the helix axis and colored as in Part c but with the C atoms

of the nucleotide pair closest to the viewer green. H atoms in

Parts b, c, and d have been omitted for clarity. [Based on X-ray

structures by the following:A-DNA, Olga Kennard, Dov

Rabinovitch, Zippora Shakked, and Mysore Viswamitra,

Cambridge University, U.K. Nucleic Acid Database ID ADH010;

B-DNA, Richard Dickerson and Horace Drew, Caltech. PDBid

1BNA; and Z-DNA,Andrew Wang and Alexander Rich, MIT.

PDBid 2DCG. Illustration, Irving Geis. Image from the Irving

Geis Collection, Howard Hughes Medical Institute. Reprinted

with permission. Model coordinates for Parts c and d generated

by Helen Berman, Rutgers University.]

See Kinemage

Exercises 17-1, 17-4, 17-5, and 17-6

JWCL281_c29_1143-1172.qxd 8/2/10 8:06 PM Page 1147

many of which could be crystallized. Consequently,

some 25 years after the Watson–Crick structure was

formulated, its X-ray crystal structure was clearly vis-

ualized for the first time when Richard Dickerson and

Horace Drew determined the first X-ray crystal struc-

ture of a B-DNA, that of the self-complementary dode-

camer d(CGCGAATTCGCG), at near-atomic (1.9 Å) res-

olution. This molecule, whose structure was subsequently

determined at significantly higher (1.4 Å) resolution by

Loren Williams, has an average rise per residue of 3.3 Å

and has 10.1 bp per turn (a helical twist of 35.5° per bp),

values that are nearly equal to those of canonical B-DNA.

However, individual residues depart significantly from

this average conformation (Fig. 29-1a, middle panel). For

example, the helical twist per base pair in this dodecamer

ranges from 26° to 43°. Each base pair further deviates

from its ideal conformation by such distortions as pro-

peller twisting (the opposite rotation of paired bases

about the base pair’s long axis; in the 1.4-Å resolution

structure, this quantity ranges from 23° to 7°) and base

pair roll (the tilting of a base pair as a whole about its long

axis; this quantity ranges from 14° to 17°).

X-ray and NMR studies of numerous other double heli-

cal DNA oligomers have amply demonstrated that the con-

formation of DNA, particularly B-DNA, is irregular in a

sequence-specific manner, although the rules specifying

how sequence governs conformation have proved to be

surprisingly elusive. This is because base sequence does not

so much confer a fixed conformation on a double helix as it

establishes the deformability of the helix. Thus, 5¿-R–Y-3¿

steps (where R and Y are the abbreviations for purines and

pyrimidines, respectively) in B-DNA are easily bent

because they exhibit relatively little ring–ring overlap be-

tween adjacent base pairs. In contrast, both Y–R steps and

R–R steps (the latter, due to base pairing, are equivalent to

Y–Y steps), and most notably A–A steps, are more rigid

because the extensive ring–ring overlap between their ad-

jacent base pairs tends to keep these base pairs parallel.

This phenomenon, as we shall see, is important for the

sequence-specific binding of DNA to proteins that process

genetic information. This is because many of these proteins

wrap their target DNAs around them, in many cases by

bending them by well over 90°. DNAs with different

sequences than the target DNA would not bind so readily

to the protein because they would resist deformation to the

required conformation more than the target DNA.

B. Other Nucleic Acid Helices

X-ray fiber diffraction studies, starting in the mid-1940s, re-

vealed that nucleic acids are conformationally variable

molecules. Indeed, double helical DNA and RNA can as-

sume several distinct structures that vary with such factors

as the humidity and the identities of the cations present, as

well as with base sequence. For example, fibers of B-DNA

form in the presence of alkali metal ions such as Na



when

the relative humidity is 92%. In this subsection, we de-

scribe the other major conformational states of double-

stranded DNA as well as those of double-stranded RNA

and RNA–DNA hybrid helices.

a. A-DNA’s Base Pairs Are Inclined to the Helix Axis

When the relative humidity is reduced to 75%, B-DNA

undergoes a reversible conformational change to the so-

called A form. Fiber X-ray studies indicate that A-DNA

forms a wider and flatter right-handed helix than does B-

DNA (Fig. 29-1, left panels; Table 29-1). A-DNA has 11.6

bp per turn and a pitch of 34 Å, which gives A-DNA an ax-

ial hole (Fig. 29-1b, d, left panels). A-DNA’s most striking

feature, however, is that the planes of its base pairs are

1148 Chapter 29. Nucleic Acid Structures

Table 29-1 Structural Features of Ideal A-, B-, and Z-DNA

A-DNA B-DNA Z-DNA

Helical sense Right-handed Right-handed Left-handed

Diameter ⬃26 Å ⬃20 Å ⬃18 Å

Base pairs per 11.6 10 12 (6 dimers)

helical turn

Helical twist 31° 36° 9° for pyrimidine–purine steps;

per base pair 51° for purine–pyrimidine steps

Helix pitch 34 Å 34 Å 44 Å

(rise per turn)

Helix rise per 2.9 Å 3.4 Å 7.4 Å per dimer

base pair

Base tilt normal 20° 6° 7°

to the helix axis

Major groove Narrow and deep Wide and deep Flat

Minor groove Wide and shallow Narrow and deep Narrow and deep

Sugar pucker C3¿-endo C2¿-endo C2¿-endo for pyrimidines; C3¿-endo for purines

Glycosidic bond Anti Anti Anti for pyrimidines; syn for purines

Source: Mainly Arnott, S., in Neidle, S. (Ed.), Oxford Handbook of Nucleic Acid Structure, p. 35, Oxford University Press (1999).

JWCL281_c29_1143-1172.qxd 7/8/10 8:39 PM Page 1148

tilted 20° with respect to the helix axis. Since its helix axis

passes “above” the major groove side of the base pairs (Fig.

29-1b, d, left panels) rather than through them as in B-

DNA,A-DNA has a deep major groove and a very shallow

minor groove; it can be described as a flat ribbon wound

around a 6-Å-diameter cylindrical hole. Most self-comple-

mentary oligonucleotides of

10 base pairs, for example,

d(GGCCGGCC) and d(GGTATACC), crystallize in the

A-DNA conformation. Like B-DNA, these molecules ex-

hibit considerable sequence-specific conformational varia-

tion although the degree of variation is less than that in

B-DNA.

A-DNA has, so far, been observed in only three biolog-

ical contexts: at the cleavage center of topoisomerase II

(Section 29-3Cd), at the active site of DNA polymerase

(Section 30-2Ae), and in certain Gram-positive bacteria

that have undergone sporulation (the formation, under en-

vironmental stress, of resistant although dormant cell types

known as spores; a sort of biological lifeboat). Such spores

contain a high proportion (20%) of small acid-soluble

spore proteins (SASPs). Some of these SASPs induce B-

DNA to assume the A form, at least in vitro. The DNA in

bacterial spores exhibits a resistance to UV-induced dam-

age that is abolished in mutants that lack these SASPs.This

occurs because the B S A conformation change inhibits

the UV-induced covalent cross-linking of pyrimidine bases

(Section 30-5Aa), in part by increasing the distance be-

tween successive pyrimidines.

b. Z-DNA Forms a Left-Handed Helix

Occasionally, a seemingly well-understood or at least fa-

miliar system exhibits quite unexpected properties. Over

25 years after the discovery of the Watson–Crick structure,

the crystal structure determination of the self-complemen-

tary hexanucleotide d(CGCGCG) by Andrew Wang and

Alexander Rich revealed, quite surprisingly, a left-handed

double helix (Fig. 29-1, right panels; Table 29-1). A similar

helix is formed by d(CGCATGCG). This helix, which has

been dubbed Z-DNA, has 12 Watson–Crick base pairs per

turn, a pitch of 44 Å, and, in contrast to A-DNA, a deep mi-

nor groove and no discernible major groove (its helix axis

passes “below” the minor groove side of its base pairs; Fig.

29-1b,d, right panels). Z-DNA therefore resembles a left-

handed drill bit in appearance. The base pairs in Z-DNA

are flipped 180° relative to those in B-DNA (Fig. 29-2)

through conformational changes discussed in Section 29-

2A. As a consequence, the repeating unit of Z-DNA is a

dinucleotide, d(XpYp), rather than a single nucleotide as it

is in the other DNA helices. The line joining successive

phosphorus atoms on a polynucleotide strand of Z-DNA

therefore follows a zigzag path around the helix (Fig.29-1a,c,

right panels; hence the name Z-DNA) rather than a

smooth curve as it does in A- and B-DNAs (Fig. 29-1a,c,

left and middle panels).

Fiber diffraction and NMR studies have shown that

complementary polynucleotides with alternating purines

and pyrimidines, such as poly d(GC) ⴢ poly d(GC) and poly

Section 29-1. Double Helical Structures 1149

B-DNA

Z-DNA

B-DNA

(or

A-DNA)

3' 5' 3' 5'

5' 3' 5' 3'

Figure 29-2 Conversion of B-DNA to Z-DNA. The conversion,

here represented by a 4-bp DNA segment, involves a 180°

flip of each base pair (curved arrows) relative to the sugar–

phosphate chains (compare the base pair orientations of B- and

Z-DNAs in Fig. 29-1b, d). Here, the different faces of the base pairs

are colored red and green. Note that if the drawing on the left is

taken as looking into the minor groove of unwound A- or B-DNA,

then in the drawing on the right, we are looking into the major

groove of the unwound Z-DNA segment. [After Rich, A., Nord-

heim,A., and Wang,A.H.-J., Annu. Rev. Biochem. 53, 799 (1984).]

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d(AC) ⴢ poly d(GT), take up the Z-DNA conformation at

high salt concentrations. Evidently, the Z-DNA conforma-

tion is most readily assumed by DNA segments with alter-

nating purine–pyrimidine base sequences (for structural

reasons explained in Section 29-2A). A high salt concentra-

tion stabilizes Z-DNA relative to B-DNA by reducing the

otherwise increased electrostatic repulsions between clos-

est approaching phosphate groups on opposite strands

(8 Å in Z-DNA vs 12 Å in B-DNA). The methylation of

cytosine residues at C5, a common biological modification

(Section 30-7), also promotes Z-DNA formation since a

hydrophobic methyl group in this position is less exposed

to solvent in Z-DNA than it is in B-DNA.

Does Z-DNA have any biological function? Rich has

proposed that the reversible conversion of specific seg-

ments of B-DNA to Z-DNA under appropriate circum-

stances acts as a kind of switch in regulating genetic expres-

sion, and there are indications that it transiently forms

behind actively transcribing RNA polymerase (Section 31-

4As). It was nevertheless surprisingly difficult to prove the

in vivo existence of Z-DNA.A major difficulty was demon-

strating that a particular probe for detecting Z-DNA, for

example, a Z-DNA-specific antibody, does not in itself

cause what would otherwise be B-DNA to assume the Z

conformation—a kind of biological uncertainty principle

(the act of measurement inevitably disturbs the system be-

ing measured). However, Rich has discovered several pro-

teins that specifically bind Z-DNA, including a family of Z-

DNA-binding protein domains named Z. The existence

of these proteins strongly suggests that Z-DNA does, in

fact, exist in vivo.

The X-ray structure of the 81-residue Z domain from

the RNA editing enzyme ADAR1 (Section 31-4As) in com-

plex with d(TCGCGCG) has been determined (Fig. 29-3a).

The CGCGCG segment of this heptanucleotide is self-com-

plementary, and therefore forms a 2-fold symmetric, 6-bp

segment of Z-DNA with an overhanging dT at the 5¿ end

of each strand (although these dT’s are disordered in the

1150 Chapter 29. Nucleic Acid Structures

(a)

(

)

Figure 29-3 Structures of DNA in complex with ADAR1 Z␣.

(a) X-ray structure of the complex of two Z domains with

Z-DNA consisting of a duplex of the self-complementary

hexamer d(CGCGCG) as viewed along its 2-fold axis of

symmetry. The Z-DNA is shown in stick form with its backbones

red and its remaining portions pink.The Z domains are drawn

in ribbon form with helices blue and sheets light green. Note that

each Z domain contacts only one strand of the Z-DNA.

(b) X-ray structure of a junction between Z-DNA and B-DNA.

The complex consists of d(GTCGCGCGCCATAAACC) and

d(ACGGTTTATGGCGCGCG) in complex with four Z

domains (not shown here for the sake of clarity).The DNA

forms a duplex with a 2-nucleotide overhang at each end in

which the lower 8 nucleotide pairs form Z-DNA, the upper 6

nucleotide pairs form B-DNA, and the A and T nucleotides at

the interface between these two DNA segments have been

extruded from the double helix.The DNA is drawn in space-

filling form with C gray, N blue, O red, and P yellow. The white

lines connect adjacent P atoms in the same polynucleotide,

thereby showing the zigzag pattern of the left-handed Z-DNA

and the smoother pattern of the right-handed B-DNA. [Courtesy

of Alexander Rich, MIT. PDBids 1QBJ and 2ACJ.]

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