Quadruplexes - Biochemistry

CHAPTE R 4

G- Quadruplexes

As long ago as 1962 David R. Davies realized that four guanine molecules could fit together in a planar hydrogen-bonded structure, and he proposed that such structures might form naturally in guanine-rich sections of DNA. Figure 4.32 shows schematically how four guanines can link to form a G-quartet (panel A), and how a single strand of guanine-rich DNA can fold to form a G-quadruplex (panel B), in the example shown consisting of three G-quartets. A G-quadruplex structure can form from one DNA strand, as shown, or from as many as four strands. G-quadruplexes actually form in living cells and exist in telomeres, spe-cial sequences at the ends of linear eukaryotic chromosomes (see Chapter 25).

Figure 4.32c shows the likely folding pattern of a human telomere. More recent studies show that G-quadruplexes exist in the transcriptional control sites, or pro-moters, of several biologically important genes, including the oncogene cMYC (Chapter 23). Current efforts are aimed at targeting such structures with anti-cancer drugs.

An Unexpected Primary Structure Modification: DNA Phosphorothioation All of the unconventional DNA structures described thus far affect secondary and/or tertiary structure, with the phosphodiester link being invariant. Accordingly, there was great surprise in 2007 when Peter Dedon’s group at MIT described an enzyme system in bacteria that converts a phosphate group in DNA to a

FIGURE 4.30

Base-pairing in one type of DNA triple helix. Both normal Watson–Crick pairing and the unusual Hoogsteen pairing occur on the same A residue.

Hoogsteen pairing

Watson–Crick pairing C′₁ C1′

C′₁

T FIGURE 4.29

A palindromic DNA sequence. A palindrome is strandwise symmetrical about a center of symmetry.

Note that in the portion in brown and blue, both blue segments read the same 5 3 , as do both brown segments. The sequence is shown in its extended and cruciform conformations. Two bases that will pair with each other in the cruciform conformation are given the same number.

¿ 4

G C C G A G T A G C T A C T C A T T

– –

C G G C T C A T C G A T G A G T A A

– – – –

– –

1 2 3 4 5 6 5′

5′ 6′ 4′ 3′ 2′ 1′

3 2

4 5 5′

1′ 3′

3′ 5′

3′

6 6′ 2′ 4′

G C C G

A G T A G

A T T

– – –

–

– C –

–––––

T C A T C

–––––

–

– – –

1 6

3 2 4

5 5′

5′

C G G

– – –

3′

1′ 3′ 6′

2′ 4′

G A G T A G

–

–––––

C T C A T C

–––––

6 3 2

4 5 5′ 1′ 3′

6′ 2′

4′

3′ T–A–A– 5′ Extended conformation

Cruciform conformation

– –

118

^{CHAPTER 4} NUCLEIC ACIDS

FIGURE 4.31

H-DNA. An H-DNA region has one all-purine (blue) and one all-pyrimidine (brown) strand, allowing it to form a triple-stranded helix by doubling back. Some segments contain both purines and pyrimidines (green). (a) The nucleotide sequence shown here is one that could give rise to H-DNA. A segment of the purine strand is shown bonded to two different seg-ments of the pyrimidine strand. (b) The bonding shown in (a) gives rise to a triple-stranded helix, shown schematically here.

G C T A A T G C G C A T G G C G G C G

G C

C G

A T

T A

A T

T A

G C

A T

G C

A T

G C

A T

G C

A T

G C

A T

G C

A T

G C

A T

G C

A T

G T

T T T

T T C⁺ C⁺ C⁺ C⁺ C⁺ C⁺ C⁺ T

G GA

A G A A

A G G A G G A

C T

G A

A T

3′ 5′

C T G A G

G C

(a) (b)

Pyr Pyr

Pur Pur

Pyr Pur

3′ 5′

Pyr Pur

FIGURE 4.32

G-quartets and quadruplexes. (a) Arrangement of bases in a G-quartet, with four Hoogsteen-bonded gua-nines surrounding a central metal ion (not shown);

(b) Folding of a single DNA strand to give a G-quadruplex, consisting in this example of three planar G-quartets.

(c) Two views of the G-quadruplex formed by the DNA sequence in human telomeres. Yellow, guanine; red, adenine; blue, thymine.

(c) J. Dai, C. Punchihewa, A. Ambrus, D. Chen, R. A. Jones, and D. Yang, Structure of the intramolecular human telom-eric G-quadruplex in potassium solution: A novel adenine triple formation, Nucleic Acids Research 35:2440–2450,

(a)

(c)

(b)

A26 A9

G T

G G A C

A G G G N

N H

N N H

N N

H H N

O O

N H

H H N

N H

O H

N H

N N N

H N H

STABILITY OF SECONDARY AND TERTIARY STRUCTURE

119

FIGURE 4.33

A phosphorothioate link in DNA, showing the cor-rect stereoisomeric form. The source of sulfur is the amino acid cysteine. Nucleotide modification occurs after nucleotide polymerization.

O O B

B O

P S⁻

O O

FIGURE 4.34

Denaturation of DNA. (a) When native (double-stranded) DNA is heated above its “melting” temperature, it is denatured (separates into single strands). The two random-coil strands have a higher entropy than the double helix. (b) At low T, G is positive and denaturation of DNA is not favored. As T increases, ⫺T S overcomes H, mak-ing G negative and denaturation favorable. The midpoint of the curve marks the “meltmak-ing” temperature, T_m, of DNA. (c) Absorption spectra of native and denatured DNA show that native DNA absorbs less light than denatured DNA, with the maximum difference occurring at a wavelength of 260 nm. This hypochromicity of double-stranded DNA can be used to distinguish between native and denatured forms. (d) The change in absorbance can be used to follow the denaturation of DNA as tempera-ture increases. An abrupt increase in absorbance, corresponding to the sudden “melting” of DNA, is seen at T_m.

⌬

⌬ Heat

Cool slowly

Denatured DNA

Denatured

Native Native DNA

1.5

1.0

Absorbance

0.5

2000

Wavelength, nm

Temperature (b)

(a)

ΔG° ( ) for denaturation Fraction denatured ( )

0 0.5 1.0

− +

Temperature (d)

(c)

T_m

Absorbance at 260 nm

1.0 1.5

0.5

T_m

260

250 300

Native Denatured

phosphorothioate, as shown in Figure 4.33. The reaction is stereospecific (note that introduction of a sulfur creates an asymmetric center about the phosphorus atom).

So far the biological function of this modification is unknown, but it might confer resistance to some foreign invaders, such as bacteriophages, which could degrade unmodified DNA. Recent sequence analysis shows that phosphorothioate nucleotides are clustered in many bacteria, consistent with the idea that phospho-rothioation could be part of a restriction-modification system (Chapter 26).

Stability of Secondary and Tertiary Structure

The Helix-to-Random-Coil Transition: Nucleic Acid Denaturation

The major polynucleotide secondary structures (the A and B forms) are relatively stable for RNA and DNA, respectively, under physiological conditions. Yet they must not be too stable because important biochemical processes—DNA replica-tion and transcripreplica-tion, for example—require that the double-helix structure be opened up. When it extends over large regions, this loss of secondary structure is called denaturation (Figure 4.34). Competing factors create a balance between structured and unstructured forms of nucleic acids.

Two major factors favor dissociation of double helices into randomly coiled single chains. First is the electrostatic repulsion between the chains. At physiolog-ical pH, every residue on a DNA or an RNA molecule carries a negative charge on the phosphate group. Even though this charge is partially neutralized by small

120

^{CHAPTER 4} NUCLEIC ACIDS

counterions (like and ) present in the medium, a substantial net negative charge remains on each chain in the helix and tends to drive the two chains apart. Therefore, high ionic strength tends to stabilize the double helix.

A more subtle factor favoring denaturation is that the random-coil structure has a higher entropy, resulting from the greater randomness of the denatured form, with its many possible configurations. Consider Equation 3.9 on page 64 ( ln W): If a rigid double helix separates into two flexible random coils, the number of configurations accessible to the molecule greatly increases (Figure 4.34a);

therefore, the entropy increases. The free energy change in going from a regular two-stranded polynucleotide secondary structure (such as B-form DNA) to indi-vidual random-coil strands is given by the usual formula:

(4.3)

Because is positive, the term makes a negative contribution to the free energy change, favoring denaturation.

Thus, two factors favor the transition: the higher randomness of the random coil and the electrostatic repulsion between chains If the double-helical structure is to be stable under any conditions, for the unfolding reaction must be positive. Therefore, we must look for a large contribution from to compensate for the factors just mentioned. The sources of such a positive are the hydrogen bonds between the base pairs and van der Waals interactions between stacked bases. In fact, the planar bases stack upon one another in van der Waals contact. Much energy must be expended to break these bonds and interactions, and hence the total is positive.

Because and in Equation 4.3 are both positive, the sign of will change as T is increased. At low temperature, the term will be less than will be and the helix will be stable. But as the temperature is increased, will become greater than and will become negative.

Thus, at higher temperatures the double-stranded structure becomes unstable and will fall apart (Figure 4.34a).

One can follow this denaturation process by observing the absorbance of ultraviolet light of wavelength about 260 nm in a DNA solution. As mentioned on page 94, all nucleotides and nucleic acids absorb light strongly in this wave-length region. When the nucleotides are polymerized into a polynucleotide, and the bases packed into a helical structure, the absorption of light is reduced (Figure 4.34c). This phenomenon, called hypochromism, results from close inter-action of the light-absorbing purine and pyrimidine rings. If the secondary structure is lost, the absorbance increases and becomes closer to that of a mixture of the free nucleotides. Therefore, raising the temperature of a DNA solution, with accompanying breakdown of the secondary structure, will result in an absorbance change like that shown in Figure 4.34d.

The remarkable feature of this helix-to-random-coil transition is that it is so abrupt. It occurs over a very small temperature range, almost like the melting of ice into water, as described in Chapter 3. Therefore, nucleic acid denaturation is some-times referred to as a melting of the polynucleotide double helix, even though the term is not technically correct. We shall encounter similar abrupt changes in con-figuration of proteins in Chapter 6. They are always characteristic of what are called cooperative transitions. What this term means in the case of DNA or RNA is that a double helix cannot melt bit by bit. If you examine the kinds of structure shown in Figures 4.11 and 4.20, you will see that it would be very difficult for a sin-gle base to pop out of the stacked, hydrogen-bonded structure. Rather, the whole structure holds together until it is at the verge of instability and then denatures over a very narrow temperature range.

The “melting temperature” of a polynucleotide depends on its ratio. Because each G-C base pair forms three hydrogen bonds and each A-T pair only two, ⌬His greater for the melting of GC-rich

(G + C)>(A + T) (T_m)

⌬G

⌬H, T⌬S 7 0,

⌬H; ⌬G ⌬H ⌬S ⌬HT⌬S ⌬G

⌬H⌬H

⌬G

(⌬Hel 6 0). (⌬S 7 0)helix 4 coil (T⌬S)

⌬S

⌬G = ⌬H - T⌬S (helix m random coil)

S = k

Mg²⁺ K⁺, Na⁺,

AT-rich regions melt more easily than GC-rich regions.

STABILITY OF SECONDARY AND TERTIARY STRUCTURE

121

polynucleotides. The greater stacking energy of G-C pairs also contributes to the difference. The value of corresponds to the temperature at which

(see Figure 4.34b and d). Thus,

(4.4)

(4.5)

On a per-base-pair basis, is about the same for all polynucleotides, but depends on base composition, as just described. This is why increases with increasing content. Figure 4.35 shows a graph of versus percent

for a number of naturally occurring DNAs.

DNA denaturation is reversible. For example, when heat-denatured DNA is cooled, DNA duplexes can reform. The rate of cooling must be slow, allowing time for complementary strands to find one another and pair up, or renature (also called annealing). Similarly, an RNA molecule can form a duplex with a DNA of complementary base sequence, creating a DNA–RNA hybrid, consisting of one strand each of DNA and RNA. DNA–DNA renaturation and DNA–RNA hybridization are at the root of several important research techniques, as we shall see later in this book.

Superhelical Energy and Changes of DNA Conformation

The storage of energy in DNA supercoiling is analogous to the energy required to wind up (supercoil) rubber bands. That is, the first few turns are easy, but the energy required, per turn, increases as the winding gets tighter. In fact, for DNA the amount of free energy stored in supercoiling is proportional to the square of the superhelical density

(4.6)

where K is a constant. Note that is zero when the DNA is relaxed and increases with either positive or negative supercoiling. Furthermore, because the square of is involved, the energy required to add an extra turn increases as each turn is added, and it becomes about equal to the energy that one ATP can provide when This is at least one reason why superhelix density is limited to about this value in vivo. Highly supercoiled DNA has a lot of stored energy, which can be reduced by any process that decreases the superhelix density. For example, suppose we have a circular DNA molecule with negative superhelical turns. If one repeat of the DNA helix (10 base pairs) were to unwind and locally melt, this change would be the equivalent of a of Then, to compensate, one negative superhelical turn could be removed that is, some of the writhing has been compensated by local unwinding. Converting a segment of B-DNA to Z-DNA would be even more efficient, requiring less energy. Each 10 bp of Z-DNA that goes from B to Z goes from a twist of (one right-handed twist) to approximately (one left-handed twist). This single change amounts to which would allow relaxation of two negative superhelical turns ( ).

Formation of cruciform structures also relaxes superhelical DNA because every base pair put into a cruciform hairpin is essentially removed from superhe-lical strain. Likewise, H-DNA formation, which leaves part of one strand unpaired, has the same effect.

In other words, imposing high levels of superhelical torsion on a DNA mole-cule can promote any one of the following changes: local melting, Z-DNA formation, cruciform extension, formation of H-DNA regions, and quite likely

⌬W = +2T = -2,

-1 +1

(⌬T⌬W = +1);-1.

s = ;0.06.

(s = 0)

⌬Gsc

⌬Gsc= Ks²

(⌬Gsc)

(G + C) (G + C) TT_m_m

⌬H

⌬S

T_m = ⌬H

⌬S 0= ⌬H - Tm⌬S

⌬G = 0 T_m

060 20 40 60 80 100

70 80 90

T_m,°C

100 110

Guanine+ cytosine, mol %

FIGURE 4.35

Effect of base-pair composition on the denaturation temperature of DNA. The graph shows the rise in

“melting” temperature of DNA as its percent (G ⫹ C) increases.

Data from Journal of Molecular Biology (1962) 5:120, J. Marmur and P. Doty.

Energy stored in supercoiled DNA can be used to drive structural transformation.

122

^{CHAPTER 4} NUCLEIC ACIDS

formation of some other special conformations not yet discovered. Which of these will happen depends on what special sequences are present in the DNA circle under stress. For example:

• The presence of AT-rich regions, which melt more easily than GC-rich regions, may favor local melting.

• Alternate purine/pyrimidine tracts (like favor Z-DNA formation, especially if the Cs are methylated on carbon 5.

• Palindromes allow cruciform extension.

• A segment that is primarily purines in one strand and pyrimidines in the other may permit H-DNA formation.

Superhelical stress is both widespread and controlled in DNA molecules found in cells. As we shall see in later chapters, the special structures described here play diverse roles in the regulation of gene expression. The idea that genes may be turned on and off by changes in supercoiling is an intriguing one.

SUMMARY

There are two kinds of nucleic acids, DNA and RNA. Each is a polynucleotide, a polymer of four kinds of nucleoside -phosphates, connected by links between

hydroxyls and phosphates. RNA has the sugar ribose; DNA has deoxyribose.

The phosphodiester linkage is inherently unstable, but it hydrolyzes only very slowly in the absence of catalysts. Each naturally occurring nucleic acid has a defined sequence, or primary structure. Early evidence indicated that DNA might be the genetic material, but it was not until Watson and Crick elucidated its two-stranded secondary structure in 1953 that it became obvious how DNA might direct its own replication. The structure they proposed involved specific pairing between A and T and between G and C. The helix is right-handed, with about 10.5 base pairs (bp) per turn in the B form. Such a structure can replicate in a semiconservative manner, as demonstrated by Meselson and Stahl in 1958. Other forms of polynucleotide struc-tures exist, of which the most important is the A form, found in RNA–RNA and DNA–RNA double helices. In vivo, most DNA is double-stranded; some molecules are circular. Most of the circular DNA molecules found in nature are supercoiled.

Most RNA is single-stranded, but it may fold back to form hairpins and other well-defined tertiary structures.

The biological functions of nucleic acids may be briefly summarized as follows:

DNA contains stored genetic information, which is transcribed into RNA. Some of these RNA molecules act as messengers to direct protein synthesis. The messenger RNA is translated on a ribosome, using the genetic code, to produce proteins. Modern molecular biological techniques allow us to manipulate DNA to make new proteins and modify existing ones.

Supercoiling of DNA can be expressed in terms of twist (T) and writhe (W). These terms are related to the linking number (L) by To form superhelical coil-ing requires the expenditure of ATP energy, uscoil-ing an enzyme called DNA gyrase.

Gyrase is one of a class of topoisomerases; others relax supercoiled DNA.

Polynucleotides can form a number of unconventional structures, including left-handed DNA (Z-DNA), cruciforms, in some cases triple helices, and G-quadruplexes.

The secondary structures of polynucleotides can be changed in various ways. The helix can “melt,” which involves strand separation. This change is easiest for regions rich in A-T pairs. Energy stored in superhelical DNA may promote local DNA melting or changes to a variety of alternative structures, such as Z-DNA, cruciforms, or a par-ticular triple-helical structure called H-DNA.

L = T + W.

5¿

3¿ 5¿

gCGCGCG g)

REFERENCES

General

Bates, A. D., and A. Maxwell (1993) DNA Topology. Oxford University Press, New York. A clear, helpful little book.

Saenger, W. (1984) Principles of Nucleic Acid Structure. Springer-Verlag, New York. This reference provides much greater detail concerning nucleic acid structure than is given in this book.

van Holde, K. E., W. C. Johnson, and P. S. Ho (2006) Principles of Physical Biochemistry (2nd ed.). Pearson/Prentice Hall, Upper Saddle River, N.J.

Has much more on nucleic acid stability and structural transitions.

Historical

Avery, O. T., C. M. MacLeod, and M. McCarty (1944) Studies on the chemical transformation of pneumococcal types. J. Exp. Med.

79:137–158. The pioneering study that lent credence to the idea that DNA is the genetic substance.

Hershey, A. D., and M. Chase (1952) Independent function of viral pro-tein and nucleic acid on growth of bacteriophage. J. Gen. Physiol.

36:39–56. The convincing evidence that DNA is the genetic material.

Judson, H. (1979) The Eighth Day of Creation. Simon & Schuster, New York. A detailed, fascinating account of the development of modern ideas about nucleic acids.

Manchester, K. L. (2007) Historical opinion: Erwin Chargaff and his

“rules” for the base composition of DNA: Why did he fail to see the possibility of complementarity? Trends Biochem. Science 33:65–70. A fresh look at historical aspects of DNA structure.

Meselson, M., and F. Stahl (1958) The replication of DNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 44:671–682. An example of a beauti-fully designed and executed experiment.

Sayre, A. (1978) Rosalind Franklin and DNA. W. W. Norton, New York. An account of the contributions of the scientist who created the best early X-ray diffraction patterns of DNA fibers.

Watson, J. D. (1968) The Double Helix. Atheneum, New York (trade and paperback editions); New American Library, New York (paperback).

An outspoken account of the elucidation of DNA structure by one of the central characters.

Watson, J. D., and F. H. C. Crick (1953) Molecular structure of nucleic acids. A structure for deoxyribose nucleic acid. Nature 171:737–738.

Two pages that shook the world.

Specialized Papers of Importance

Bacolla, A., and R. D. Wells (2004) Non-B DNA conformations, genomic rearrangements, and human diseases. J. Biol. Chem. 279:47411–47414.

A minireview dealing with unconventional DNA structures.

Burge, S., G. N. Parkinson, P. Hazel, A. K. Todd, and S. Neidle (2006) Quadruplex DNA: Sequence, topology, and structure. Nucleic Acids Research 34:5402–5415. Chemistry and biology of this unusual DNA structure.

Castro, C. E., F. Kilchherr, D-N. Kim, E. L. Shiao, J. Wauer, P. Wortmann, M. Bathe, and H. Dietz (2011) A primer to scaffolded DNA origami.

Nature Methods 8:221–229. A recent instruction manual for creating three-dimensional DNA assemblies.

Deweese, J. E., M. A. Osheroff, and N. Osheroff (2009) DNA topology and topoisomerases. Teaching a “knotty” subject. Biochem. Mol. Biol.

Education 37:2–10. An exceptionally clearly written short review, with discussion of topoisomerases as drug targets.

Dietz, H., S. M. Douglas, and W. H. Shih (2009) Folding DNA into twisted and curved nanoscale shapes. Science 325:725–730. Careful design and annealing of synthetic oligonucleotides allows DNA to be folded into precise shapes, such as a miniature gear wheel.

Han, D., S. Pai, J. Nangreave, Z. Deng, Y. Liu, and H. Yan (2011) DNA origami with complex curvatures in three-dimensional space.

Science 332:342–346. A recent paper describing the use of synthetic DNA to make curved three-dimensional shapes, including a flask 70 nm high.

Joyce, G. F. (2002) The antiquity of RNA-based evolution. Nature 418:214–221. Thoughts about a primordial RNA world.

Khuu, P., M. Sandor, J. DeYoung, and P. S. Ho (2007) Phylogenomic analysis of the emergence of GC-rich transcription elements. Proc.

Natl. Acad. Sci. USA 104:16528–16533. Comparative DNA sequence analysis indicating that Z-DNA-forming sequences arose at specific stages in evolution.

Mardis, E. R. (2008) The impact of next-generation sequencing technol-ogy on genetics. Trends Genet. 24:133–141. A discussion of three new high-volume DNA sequencing technologies and some of their potential applications.

Sharma, J., R. Chhabra, A. Cheng, J. Brownell, Y. Liu, and H. Yan (2009) Control of self-assembly of DNA tubules through integration of gold nanoparticles. Science 323:112–116. More about the use of DNA molecules in nanotechnology.

Vologodskii, A. V., and N. R. Cozzarelli (1994) Conformational and ther-modynamic properties of supercoiled DNA. Annu. Rev. Biophys.

Biomol. Struct. 23:609–643.

Wang, L., S. Chen, T. Xu, K. Taghizadeh, J. S. Wishnok, X. Zhou, D. You, Z. Deng, and P. C. Dedon (2007) Phosphorothioation of DNA in bacteria by dnd genes. Nature Chem. Biol. 3:709–710. Surprising news about a new internucleotide link in DNA.

Wing, R. M., H. R. Drew, T. Takano, C. Brodka, S. Tanaka, K. Itakura, and R. E. Dickerson (1980) Crystal structure analysis of a complete turn of B-DNA. Nature 287:755–758. First crystallographic study of a B-DNA structure.

Wong, L., and 14 coauthors (2011) DNA phosphorothioation is wide-spread and quantized in bacterial genomes. Proc. Natl. Acad. Sci.

USA 108:2963–2968. Use of a mass spectrometric technique for sequence analysis of the phosphorothioate modification.

REFERENCES

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^{CHAPTER 4} NUCLEIC ACIDS

PROBLEMS

1. A viral DNA is analyzed and found to have the following base com-position, in mole percent:

(a) What can you conclude about the structure of this DNA?

(b) What kind of secondary structure do you think it would have?

2. Given the following sequence for one strand of a double-stranded oligonucleotide:

(a) Write the sequence for the complementary DNA strand.

(b) Suppose you knew that the strand shown above had phosphate on both ends. Using an accepted nomenclature, write the sequence so as to show this.

*3. Some naturally occurring polynucleotide sequences are palindromic;

that is, they are self-complementary about an axis of symmetry.

Such a sequence is

Show how this structure might form a double hairpin, or cruciform, conformation. Indicate the center of symmetry in the sequence and the bounds of the cruciform.

4. The E. coli genome has a superhelical density in vivo of about 0.06.

Assuming the DNA has 10.5 bp/turn, what is the expected writhing number of the E. coli genome?

*5. Given the following sequence for an RNA molecule, find a second-ary structure that will be maximally stable.

6. The largest of the double-stranded RNA molecules of cytoplasmic polyhedrosis virus contains 5150 bp (see Table 4.5). How long do you expect this molecule to be if extended?

7. A circular, double-stranded DNA contains 2100 base pairs. The solution conditions are such that DNA has 10.5 bp/turn.

(a) What is for this DNA?

(b) The DNA is found to have 12 left-handed superhelical turns.

What is the superhelix density

*8. In a supercoiled DNA, a stretch of about 20 base pairs changes from the B form to the Z form. What is the change in (a) T, (b) L, and (c) W?

9. Of the DNA molecules listed in Table 4.4, which would you expect to have the highest and lowest

10. A scientist isolates the DNA genome from a virus. She attempts to carry out a melting analysis but finds only 10% hypochromicity.

(a) Suggest an explanation for the low value.

(b) Why do you think she finds this much?

11. A particular double-stranded DNA has, under the conditions used in Figure 4.32, a melting point of 94 °C. Estimate the base composi-tion (in mole percent) of this DNA.

Tm? s?

GUCCAGCCAUUGCGUUCGCAAUGGC AGTTCAGGTACCTGAACC TCAAGTCCATGGACTTGG 5¿ACCGTAAGGCTTTAG3¿

A = 32, G = 16, T = 40, C = 12.

*12. A variant of B-form DNA has been reported to exist in the presence of ions. This form, called C-DNA, is found by X-ray diffraction to have 9¹⁄3base pairs per turn.

(a) How many base pairs are contained in one repeat of this struc-ture? How many turns in one repeat?

(b) Is C-DNA twisted more or less tightly than B-DNA?

*13. A closed circular supercoiled DNA is relaxed by treatment with topoisomerase. No matter how much enzyme is used, or how long the experiment is run, the experimenter always finds a gel elec-trophoresis pattern indicating some DNA with one, two, and three superhelical turns in addition to the relaxed (nicked) circle (see fig-ure). Suggest an explanation for this observation.

*14. The dye ethidium is a planar molecule that can intercalate into double-stranded DNA. This means that it slips between adjacent stacked base pairs. In doing so, it unwinds the DNA helix by about 26° for every ethidium bound.

(a) If ethidium were added to relaxed, closed circular DNA, would negative or positive writhing be expected? Explain.

(b) What would be the effect on writhing if the DNA were nicked in one strand?

(c) It is observed that progressive addition of ethidium to negatively supercoiled DNA has a peculiar effect: First the electrophoretic mobility decreases, but with further addition of ethidium it again increases. Explain.

*15. Explain why DNA is stable in the presence of alkali (0.3 M KOH), while RNA is quantitatively degraded to - and -nucleoside monophosphates under these conditions.

*16. Refer to Table 4.1 for the values for ionization of the four ribonucleoside -monophosphates. Select a pH value at which each of the four nucleotides has a different net charge on the molecule, and predict the direction and relative rate of migration of each nucleotide in an electrophoretic field.

5¿

pKa

3¿ 2¿

−

Nicked circle Li⁺

Dalam dokumen Biochemistry (Halaman 144-152)