Nucleic Acid Structure

• Nucleic acids form double-stranded secondary structures, with hydrogen bonds between complementary bases (G to C, and A to T or A to U), either between 2 different strands or between different parts of the same strand folding back on themselves; the base-paired sequences are complementary and antiparallel for formation of secondary structure.
  • The most important stabilizing factor for double-stranded nucleic acids is stacking interactions, which result from van der Waals and dipole-dipole interactions between adjacent base pairs in core of double helical structure, and the hydrophobic effect.
  • Double-stranded nucleic acid structures (secondary structure) can be disrupted by heat, the temperature midpoint of the transition being called "T_m", the "melting temperature".
    • higher G+C base content of a double-stranded DNA sample --> a higher T_m (greater stability of duplex structure) due mainly to stronger stacking interactions of G-C base pairs.
    • stability of duplex also influenced by pH and by salt concentration
    • Renaturation (re-formation of double helical structure from complementary single strands, either DNA-DNA or DNA-RNA) = annealing, or hybridization
• 3 double-helical conformations of nucleic acids
  • B-DNA = the double helical conformation of most DNA under physiological conditions (right-handed, intermediate diameter, tightly packed atoms in core, bases nearly perpendicular to helix axis)
  • A-DNA = double helical form favored by DNA-RNA and RNA-RNA duplexes, as well as by DNA-DNA duplexes under dehydrating conditions (right-handed, wider diameter, "hole" down the center, bases tilted much more relative to helix axis)
  • Z-DNA = double helical form adopted by some alternating pyrimidine-purine sequences at high salt concentrations (left-handed, very narrow diameter, phosphates in backbone "zig-zag")
• cellular RNA: 3 classes -- tRNA, rRNA, mRNA
  • tRNA and rRNA have extensive secondary and tertiary structure
• DNA replicated semiconservatively -- in the process of transcription, each strand of the DNA duplex is copied into a complementary daughter strand.
• Nucleases (exo- and endo-) catalyze hydrolysis of phosphodiester bonds
  • Restriction endonucleases -- highly sequence specific, indispensible tools in recombinant DNA work.
• DNA supercoiling is biologically very important, e.g., for compaction of DNA for packaging in the cell, and for easier strand separation for replication and transcription.
  • Cellular DNA is maintained in an underwound state (more bp/turn than relaxed DNA, which has about 10.4-10.5 bp/turn)
  • Structural strain (sub-optimal base stacking) in duplex DNA resulting from having fewer turns than required for relaxed DNA is relieved by
    • partial strand separation, or
    • supercoiling (winding of double helix past its own axis)
  • Linking number (Lk) is the number of helical turns in a closed circular DNA molecule (or DNA with its ends "held" so it can't change the number of helical turns).
  • Lk = number of bp (length) / (actual number of bp/turn)
  • Topoisomers = DNA molecules identical to each other in sequence but differing in a topological property like Lk
  • Enzymes that change Lk = topoisomerases
  • DNA in eukaryotic chromosomes is compacted at the "lowest" hierarchical level by having the double helix supercoiled around proteins (histones) in nucleosomes.

Nucleic Acid Structure 

Primary Structure

• primary structure (as for proteins) = sequence of nucleotides along polymer
• directionality in sequence ("polarity" of sequence): 5¢ end (hydroxyl or phosphate ester on 5'C at one end) and a 3¢ end (hydroxyl or phosphate ester on 3'C at other end)
• Convention: nucleic acid sequences written from the free 5¢ hydroxyl (or phosphate) and ending at the free 3¢ hydroxyl (or phosphate), so sequence is written from 5' --> 3' left to right unless otherwise stated.
• In this convention, phosphodiester linkages run from 3' C of one nucleotide to 5' C of next nucleotide.

Secondary Structure

• Secondary structure of DNA = double-stranded structure with bases hydrogen-bonded to complementary bases on another strand or sometime doubling back to "base-pair" with a complementary sequence within the same strand
  • The well-known double helix of B-DNA is the most common form of DNA secondary structure under physiological conditions.
    
    
• What did Watson and Crick know when they were deducing this structure?
  1. They had an x-ray diffraction pattern of fibers of DNA, from data collected in the laboratory of Maurice Wilkins by Rosalind Franklin (taken by Wilkins without Franklin's permission!), and Crick knew enough about X-ray diffraction to be able to discuss it with Wilkins and analyze it himself, with different conclusions from Franklin's.
     • Fiber diffraction patterns do not contain the molecular information found in single crystal diffraction patterns, but the fiber diffraction patterns and the properties of the fibers provided the essential information to deduce the double helix structure:
       • The diffraction pattern showed the presence of a helix.
       • The spacing between the spots showed that there are 10 bases per turn of the helix.
       • The density of the fibers indicated two molecules of DNA per helix.
  2. It had been reported by Edwin Chargaff that DNA always contained equal molar amounts of G and C (G=C), and equal molar amounts of A and T (A=T), so (G+A) = (C+T), i.e., [purines]=[pyrimidines] in DNA.
     • Chargaff didn't know why those molar ratios held.
     • Chargaff had samples of DNA from a variety of organisms, which had a variety of different (G+C)/(A+T) ratios, but no DNA samples from any single-stranded DNA viruses!
     • Watson and Crick knew "Chargaff's Rules", which helped them to concentrate on the correct complementary base relationships in their model building.
  3. An office-mate of Crick's had deduced the correct tautomeric forms of the bases in DNA, which affected their hydrogen bonding possibilities.
     • Knowing that the keto forms of the bases predominated was a great help in considering alternative ways for bases to hydrogen bond to each other.
• Watson and Crick proposed that the double helix was stabilized by hydrogen bonding between bases on the opposite strands: A = T and G = C (bpair).

A-T base pair	G-C base pair

• Watson-Crick model:
  • hydrophilic sugar-phosphate backbones are on outside, in contact with water
  • hydrophobic bases are stacked essentially perpendicular to axis of helix on the inside.
  • G on one strand forms 3 hydrogen bonds with C on other strand; A on one strand forms 2 hydrogen bonds with T on other strand.
• The two strands of DNA are complementary in primary structure and run in the opposite directions, i.e., they are antiparallel. 
• Although the bases are on the inside, they can be approached (e.g., by DNA-binding proteins) through two deep spiral grooves called the major and minor grooves. This model also accounted for the fact that in the composition of DNA the % A=%T and %G=%C.

Stability of the DNA Duplex

• Process: strand separation ("melting" of the duplex): double-stranded DNA <==> 2 single strands
• DNA duplex (double-stranded structure) favored by:
  • MAIN STABILIZING FACTOR = base stacking - a combination of
    • the hydrophobic effect (an entropic effect, getting bases out of contact with H₂O), and
    • van der Waals and dipole-dipole interactions (enthalpic effects)
  • Hydrogen bond formation between base pairs is not nearly as important as stacking interactions in stabilizing double stranded structure.
    Why aren't hydrogen bonds an important factor in determining the duplex/single strands equilibrium position?
    However, would you expect a hydrogen bond from a base N-H to an O= to be stronger if the O= is part of another base structure in the core of the double helix, or if the O= is the oxygen of water out in solution?
    
• Separate single strands ("melting") is favored by:
  • having less electrostatic repulsion of the backbone phosphates than in duplex
  • conformational entropy (one molecule --> 2 molecules, and also more freedom of single strands to adopt different conformations in solution)
  • hydrogen bond formation with water
• Although base pairing by hydrogen bonding doesn't play a big role in stabilizing the duplex structure, it provides all the specificity required for the complementarity of the 2 strands, essential for the processes of DNA replication and transcription.

Double Helical Structures 

• (see also Nelson & Cox Fig. 10-18) Different structures of double-stranded DNA result from:
  • different possible conformations of the 2-deoxyribose (C-3' endo vs. C-2' endo)
  • rotation about the bonds in the phosphodeoxyribose backbone
  • free rotation about the C1'-N-glycosyl bond
• B-DNA structure (B_DNA)
  • "Watson and Crick" double helix, the form favored for random-sequence DNA under physiological conditions
  • right-handed
  • Strands are complementary in nucleotide sequence: where one strand has a T, it's base-paired (by hydrogen bonds) to an A on the other strand; if first strand has a G, it's base-paired to a C on the other strand.
  • Strands are antiparallel: if 1 strand runs 5'-->3' left to right, its partner runs 3'-5' left to right.
  • Major groove is wide and deep; minor groove is narrow and deep.
  • tightly packed atoms in core (along helix axis), no hole
  • X-ray structure of DNA basically confirmed the double helix model, but showed that the model was an oversimplification:
    • Actual molecular structure showed many local variations and distortions from idealized structure.
    • DNA secondary structure is not rigid, but flexible, and depends on the exact nucleotide sequence and can be changed by interactions with proteins or other molecules.
• A-DNA structure (A_DNA)
  • favored for DNA-DNA duplex under dehydrating conditions
  • right-handed, double-stranded (complementary strands)
  • favored under physiological conditions for RNA-RNA ( (A_RNA) or RNA-DNA duplexes, because the 2¢ hydroxyl of ribose sterically inhibits formation of the B conformation
  • Strands antiparallel and complementary in sequence
  • Major groove narrow and deep; very little minor groove (not much of a "groove" -- wide and shallow)
  • wider diameter than B-DNA, with hole down the helix axis
• Z-DNA structure (Z_DNA)
  • left-handed form of double-stranded DNA (complementary strands)
  • backbone phosphates "zig-zag"
• favored by alternating purine-pyrimidine sequences, and high salt concentrations (which minimize the electrostatic repulsion between backbone phosphates)
• Strands antiparallel and complementary in sequence
• almost no major groove (flat); minor groove narrow and deep
• atoms very tightly packed
• physiological role uncertain -- does occur in short tracts in vivo in both prokaryotes & eukaryotes, and may have something to do with regulation of expression of some genes, or in genetic recombination
• Comparison of the three forms of the DNA double helix (see also Fig. 10-19 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000))
  The images are for DNA molecules containing 12 base pairs; all 3 helix axes are vertical.
  

  	A-DNA	B-DNA	Z-DNA
  Helical sense	right handed	right handed	left handed
  Diameter	~26 Å	~20 Å	~18 Å
  Base pairs/turn	11	10.5	12
  Helix rise/base pair	2.6Å	3.4Å	3.7Å
  Base tilt normal to helix axis	20o	6o	7o
  Side View
  End View

• In some organisms the DNA is circular (no free 3¢ or 5¢ ends) and in others linear.
• The DNA molecules in eukaryotes are immense
  • The DNA from one Drosophila melanogaster (fruit fly) has a molecular weight of 4 x 10¹⁰ g/mol (~6.5 x 10⁷ base pairs) and would be 2 cm long if fully extended (length = 0.34 nm x number of base pairs).
  • The total DNA from all the cells in one human would extend to the sun and back 50 times!
  • Obviously, these dimensions exceed the size of the organisms and DNA in the cells must be highly condensed (see below).

DNA replication is semiconservative.

• Copying mechanism involves unwinding of the two strands of a parental DNA duplex, with each strand serving as the template for synthesis of a new strand, complementary to and wound about the parental strand.
• At each base of new strand, the complementary base to the parental strand is present and is positioned during polymerization by base pairing.
• Expression of the genetic information in DNA involves transcription of the DNA sequence into RNA (messenger RNA, mRNA), then translation of the mRNA sequence into the amino acid sequence of the protein.
• In formation of the mRNA, complementary base pairing between ribonucleotides and the DNA sequence is involved.
• In the formation of proteins, each amino acid is specified by a sequence of the three bases (the codon) to which a transfer RNA, which carries the activated amino acid, binds.

DNA Denaturation/Renaturation

• Fig. 10-29 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000): Reversible denaturation ("melting") and annealing (renaturation) of DNA

Although the double helix is relatively stable under physiological conditions, the loss of secondary structure, denaturation, can be induced by: enzymes (e.g., RNA polymerase; helicases) extremes of pH Negative charges on phosphate groups can be partially neutralized by ions or proteins, reducing effect of electrostatic repulsion. Change in concentration of these "charge neutralizing" factors at high or low pH can lead to chain separation. chemicals, such as formamide, which decrease the base stacking energy of the double helix temperature (due to the fact that the entropy of the denatured state (random coil) is higher than that of the double helical state)

• free energy change for double helix --> random coil transition ("melting") is DG = DH - TDS.
  • For the double helix --> random coil transition,  DH_{electrostatic (relief of charge repulsion)} is negative and DS_{conformational} is positive, both favorable for strand separation/unfolding.
  • Therefore, in order for the double helix to be stable under any conditions there must be an unfavorable positive DH in the double helix --> random coil transition that favors the double helical state.
  • This positive DH in going from double helix to random coil individual chains arises from disruption of van der Waals and dipole-dipole interactions between bases, which is the major enthalpic factor favoring the double helix. (The hydrophobic effect also contributes an entropic factor favoring base stacking.)
  • At physiological temperature, magnitude of positive DH from loss of stacking interactions between the bases must be greater than DH_{electrostatic} - TDS_{conformational} in order for the helix to be stable.
  • However, sign of DG for will change from + to - as temperature increases due to dominance of the conformational entropy term.
  • Thus the double helix --> random coil transition will be favored at higher temperature.
• Extent of denaturation as temperature increases can be followed at 260 nm because absorbance of the bases is less when stacked in the helix than in the random coil state.
  (This increase in absorbance upon "un-stacking" the bases is called the "hyperchromic effect"; the reverse, decrease in absorbance when double helix forms and bases stack, is called the "hypochromic effect".)

Steepness of melting curve suggests that melting is a highly cooperative process (see below) - the whole DNA molecule is either in the helix state or the random coil state.. Midpoint temperature of the transition = TM Note that curve for reformation of the double helix (renaturation, annealing) is different (hysteresis) than the melting curve, and that the annealing temperature TA is lower than the TM.

Melting temperature TM, depends on both G+C content of DNA and salt concentration of solution. Recall that energy of base stacking interaction between a G-C base pair and either a G-C or A-T base pair is stronger than the interactions between A-T base pairs. Thus higher G+C content leads to higher duplex stability. High ionic strength shields electrostatic repulsion of the phosphate groups in the backbone.

Models for melting and annealing of DNA. Melting starts at ends or internally at regions rich in AT base pairs. As temperature increases, the melted regions extend until the two strands separate. Annealing is a bimolecular process, but there are competing processes in which both intrastrand and interstrand base pairs can form, ultimately leading to the stable duplex.

Nucleic Acids Hybridize by Base Pairing

• A crucial property of the double helix: ability to separate the two strands without disrupting covalent bonds.
• This makes it possible for the strands to separate and reform under physiological conditions.
• Specificity of the process is determined by complementary base pairing.

Hybridization refers to formation of double-stranded nucleic acids. often used to describe formation of a DNA-RNA hybrid, as illustrated here. Annealing of DNA is also hybridization. Hybridization also critical for: The Polymerase Chain Reaction (PCR). Southern Blotting (named for Ed Southern, who developed the method), which involves DNA-DNA hybridization (Southern). Northern Blotting, which involves DNA-RNA hybridization. Assaying DNA MicroArrays (MicroArray)

RNA - Secondary and Tertiary Structure

• RNA is synthesized by copying DNA, with complementary sequence.
• RNA molecules have complex 3-dimensional structures.
  • Single strands without self-complementary sequences tend to assume right-handed helical conformation, with extensive base-stacking of adjacent bases within the single strand.
  • RNA forms extensive secondary structure (A-form duplexes -- hairpins, stems & loops) from pairing of self-complementary sequences within the molecule, and tertiary folding in which all kinds of noncovalent bonds stabilize the folded structure.
    
• 3 major types of RNA
  • transfer RNA (tRNA)
  • ribosomal RNA (rRNA)
  • messenger RNA (mRNA).
  • Other minor forms of RNA are important for functions such as splicing and telomere synthesis.
• Transfer RNA (tRNA)
  • tRNAs small, single-stranded (73-94 nucleotides) - function in protein synthesis as "adaptors" that "read" the mRNA codons (3-base sequences) and bring to the ribosome the correct amino acid for a given codon
  • Single-stranded DNA and RNA have a tendency to adopt a random coil structure. However, such single-stranded molecules contain regions of self-complementary sequences where the molecule can form double-stranded stems ( (tRNA)
  • tRNA contains chemically modified bases, e.g. hypoxanthine, pseudouracil, dihydrouracil, and 7-methylguanine

• Ribosomal RNA (rRNA)
  • ribosome = a subcellular organelle involved in protein synthesis
    • consists of a large and a small subunit (each consisting of both RNA and multiple proteins)
    • rRNA molecules are an integral part of the structures of both subunits
      • rRNA: single stranded, but has a complex secondary structure, as shown below for 16S rRNA from E. coli (ladder-like structures are hydrogen bonded base pairs)
      • rRNA also contains chemically modified bases

Berg, Tymoczko & Stryer, Biochemistry, 5th ed., 2001: Fig. 29.17: Secondary and tertiary structures of 16S rRNA from E. coli (from X-ray crystallographic determination)

Protein-rRNA complexes of the large (50S) and small (30S) subunits of the E. coli ribosome (from a model for the ribosome constructed using cryo-electronmicroscopy) I.S. Gabashvili et al., Solution Structure of the E. coli 70S Ribosome at 11.5 Å Resolution, Cell, 100, 537-549 (2000))

• (from Berg, Tymoczko & Stryer, Biochemistry, 5th ed., 2001) Fig. 29.16: Tertiary structure of E. coli 70S ribosome (from X-ray crystallographic studies)

Proteins in red (50S subunit) and blue (30S subunit), rRNA molecules in yellow (23S rRNA), orange (5S rRNA), and green (16S rRNA)

 Messenger RNA (mRNA)

• produced (process = transcription) by enzymatic synthesis of an RNA molecule complementary to a protein-encoding strand of DNA (gene)
• the template for protein synthesis
• may assume an A-type double helix or a single-stranded structure.
• does not contain chemically modified bases.

Cleavage of DNA and RNA Polymers

Chemical Stability

RNA is susceptible to alkaline hydrolysis because of presence of free 2'-OH on ribose, as shown in this diagram. DNA is stable to alkaline hydrolysis, because it lacks the free 2'- OH on the ribose.

Nucleases

• Nucleases = enzymes that catalyze hydrolysis of the phosphodiester bonds in nucleic acids
  • Some are specific for DNA (DNases)
  • others specific for RNA (RNases)
  • still others show no specificity.
• Exonucleases remove nucleotides from the ends, either from the 5^'- or 3^'- ends.
• Endonucleases hydrolyze internal phosphodiester bonds.

Restriction Enzymes

• Viruses that infect bacteria = bacteriophages (or "phages"), which consist of protein and nucleic acid.
• Some phage that grow well in one strain of bacteria are often unable to grow well in other strains of bacteria.
• Phage do not grow because their DNA molecules are cleaved and degraded by enzymes of the host bacterial cell, a defense mechanism of the host against foreign DNA.
• Degrading DNA destroys ability of phage to grow and is responsible for the pattern of growth restriction, hence the bacterial enzymes are called restriction enzymes (restriction ENDONUCLEASES).
• Restriction enzymes, of which now more than 1000 are known, are sequence-specific. For example, EcoRI (from E. coli) is specific for the sequence (5')GAATTC in double stranded DNA.
  • What is the sequence of the complementary strand? (3') CTTAAG
  • What is the sequence of the complementary strand for these 6 nucleotides read in 5'--> 3' direction?
  • What is the term used to describe sequences (DNA) that read the same in both directions (when both strands are being read in the 5'-->3' direction)?
• Why doesn't the enzyme EcoRI digest (cleave at all the EcoRI-specific sequences) E. coli's OWN DNA, so the cell commits suicide?
  • Restriction enzymes in a bacterial cell are just half of a system known as a "restriction-modification" system
    • A strain that makes a specific restriction endonuclease also makes a DNA-modifying enzyme with the same sequence specificity as the restriction enzyme.
    • The chemical modification of a base on either strand (or both) of DNA protects BOTH strands from cleavage by the restriction enzyme.
    • e.g., E. coli strains that make EcoRI also make a DNA methylase, which introduces a CH₃-group onto the 3rd base (a C) from the 5' end of each strand; with the base methylated on even one strand the host DNA is protected from the restriction enzyme, while invading viral DNA, unmethylated, is cleaved.
• For more details look at these animations (restriction enzymes).
• Restriction enzymes are also used in DNA fingerprinting.

Tertiary Structure of DNA

• Because DNA is such a long molecule, its tertiary structure is very complex. However, we can discuss some known structures.

DNA Supercoiling (see Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000, pp. 915-923

• What is supercoiling?
  • a "coiled coil" of DNA, a level of coiling above the winding of the 2 helices around each other
  • The axis of the double helix bends and twists.
  • B-form DNA with no supercoiling ("relaxed" DNA) has about 10.5 bp (base pairs)/turn. (Actual number of bp/turn varies slightly with local sequence.)
  • Having < 10.5 bp/turn ("overwound" DNA) or > 10.5 bp/turn ("underwound" DNA) --> STRAIN, due to less than optimal base stacking.
  • SOLUTION: DNA supercoiling -- 2 biological functions:
    • compaction of DNA
    • easier strand separation for replication & for transcription

• 2 situations:
  • If start with relaxed DNA (of a certain length and certain number of turns, with 10.5 bp/turn) and partially separate strands (e.g., in replication or transcription), DNA would have < 10.5 bp/turn -- same number of turns would be spread over fewer bp of DNA --> fewer bp/turn.
    • DNA with fewer than 10.5 bp/turn = "overwound" (
    • This is a strained situation, (higher free energy form, less stable than relaxed DNA, due to less than optimal base stacking)
    • Partial strand separation for DNA that's relaxed to start with is unfavorable (DG > 0).

  • Fig. 24-11 (Nelson & Cox, Lehninger Principles of Biochermistry, 3rd ed., 2000): Supercoiling induced by separating the strands of a helical structure

  • If start with underwound DNA ( > 10.5 bp/turn, so DNA is strained, due to less than optimal base stacking), and partially separate the strands, what's the result?
    • Partial strand separation --> fewer bp/turn, so partial strand separation for underwound DNA --> closer to 10.5 bp/turn
    • Partial strand separation thus relieves some of the strain, so partial strand separation for DNA that's underwound to begin with is a favorable process (DG < 0).
  • Cellular DNA is kept about 5%-7% underwound, for easier strand separation and also for compaction.
  • 2 ways for underwound DNA ( > 10.5 bp/turn) to relieve strain:
    • If no strand separation, strain is relieved by supercoiling: helix bends and twists to achieve better base stacking orientation despite having too many bp/turn.
    • If there IS some strand separation, strain is relieved by having fewer bp/turn, closer to the optimal 10.5.
  • REMINDER: Supercoiling can only be maintained if strands can't unwind, and number of turns can't change.
    • These conditions result either from
      • DNA being a closed circle, or
      • Proteins bind so strands can't rotate around each other.

   

• The DNA of bacteria, mitochondria, plastids and some viruses exists as a closed circle. 
• Circular DNA can have a twisted or coiled appearance, as seen in the electron microscope images below (from A. Kornberg DNA Replication, pg. 29, W.H.Freeman, 1980) (Fig. 24-12 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000): Relaxed and supercoiled plasmid DNAs.) 
• It is clear that supercoiling causes the DNA to assume a more compact structure:
  • Molecule at left is relaxed; degree of supercoiling increases from left to right.
    
  

• A closed circle of DNA can be formed by joining the ends of linear DNA together as shown below for a 260 bp DNA.

• In both linear form and circular form, the two DNA strands cross each other 25 times. 
  • LINKING NUMBER (Lk) = # of helical turns in a closed circular DNA molecule = total # of bp / (actual # of bp/turn).
    WHAT ARE THE "UNITS" of Lk?
    (They're never stated, just understood, but remembering the "unit"s might help you understand the concept or do calculations.)
    
  • Lk is invariant no matter how the circle is twisted. 
  • The circle above is not supercoiled and is called a relaxed circle (260 bp/25 turns = 10.4 bp/turn, very close to "theoretical" B-DNA value).
  • For closed circular DNA, Lk has to be an integer. (WHY?)
    
    
• Supercoiling is induced if the DNA is unwound before (or after) the circle is made, as shown below.

• In forming this underwound circle, we have changed the linking number from 25 to 23. 
• This DNA circle is unstable because the DNA duplex has been disrupted -- there are now too few turns for the unchanged number of bp, i.e., too many bp/turn (260 bp/23 turns = 11.3 bp/turn).
• Underwound molecule has lost 2 turns relative to the relaxed form, and (-2 turns) / 25 turns (the relaxed value) = - 0.08, so this molecule is 8% underwound.
• The duplex structure can be stabilized (bases can stack properly at closer to 10.4 or 10.5 bp/turn) if the circle twists into a supercoil as shown below, introducing 2 "supercoil" turns.

 

• An alternative way to relieve strain would be to partially separate the strands
  • If the 23 turns were confined to only about 240 bp of double helix, and the other 20 base "pairs" were actually strand-separated (as in drawing above where the 2 turns had been removed), the duplex part of the structure would be stable, with about 10.4 bp/turn.
    
    
• Identical DNA molecules differing only in linking number = TOPISOMERS.
• Enzymes that increases or decrease the extent of DNA underwinding = topoisomerases
  • very important in replication and DNA packages

• We now need two additional topological terms to describe our supercoiled DNA molecule: 
  • TWIST, Tw
  • WRITHE, Wr
  • Twist = the number of complete revolutions that one strand makes about the duplex axis.   
    • For B-DNA the twist is the number of base pairs divided by 10.4 (the number of base pairs per turn of the B-DNA double helix).
    • For this 260 bp DNA, Tw = 25. 
  • Writhe is the number of times the helix axis crosses over itself (coiling of the helix axis). 
    • The helix of our supercoiled 260 bp molecule makes two right-handed crosses over itself, so Wr = -2. 
    • Because Lk is constant for any circle, for every double helical twist added, DTw, there must be an equal and opposite supercoil twist, -DWr. 
    • The usual value of Wr is negative, which means the supercoil is right-handed.
  • The topological state of a DNA molecule is described by the equation: Lk = Tw + Wr. 
    • Thus for our 260 bp supercoiled molecule: 23 = 25 + (-2). 
    • If one of the three topological terms is changed, the other terms must also change so that: DLk = DTw + DWr.

  Fig. 24-17 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000): Ribbon model illustrating twist and writhe

Importance of supercoiling

1. Practical:  gel separation.  Supercoiled DNA moves faster on a gel (see below).
2. Biological:  The topological state of the DNA is critical for its biological function.
3. Packing of DNA:  Supercoiled DNA is more compact.

Supercoiling is regulated enzymatically

• Topoisomerase I relaxes supercoils by increase linking number in increments of 1.
• Gyrase induces supercoiling using ATP to decrease linking number in increments of 2.
• topoisomers = DNA molecules that are identical to each other in terms of sequence but differ in a topological property such as linking number
• Figure below shows effect of topoisomerase I on the highly supercoiled DNA of a virus. Enzyme introduces single strand break into the DNA duplex, passes other strand through break and reseals break.  Net effect: increase Lk and reduce Wr by +1.  When Lk = Tw, circle is relaxed.

The higher the degree of supercoiling, the more compact the molecule, so the faster it migrates by electrophoresis on the gel. The bands differ in Lk from adjacent band by 1. Relaxed DNA is the least compact form and migrates the most slowly. Agents that intercalate between the bases, such as ethidium bromide, unwind the DNA duplex and cause supercoiling (ethidium).

Chromosomes

• Eukaryotic DNA is packaged into highly condensed structures called chromosomes. Below are two views of chromosome organization.
  (Left figure has chromosome at top, with descending hierarchy of molecular structure to the DNA double helix at the bottom; right figure has double helix at top, with ascending levels of chromatin structure culminating in chromosome at bottom.)

 

 Nucleosomes

• Nucleosome formation in eukaryotic chromatin ("beads on a string" structure) -- see figure below:

146 bp of DNA wraps (coils) 1.8 times around a set of proteins (several different histones, which are highly positively charged) = one "bead", which together with about 56 bp of "linker" DNA to the next unit, = a nucleosome. The DNA in the "beads" has a large writhe, forming a solenoidal supercoil. (Nucleosome).

Fig. 24-24 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000): Structure of a nucleosome core particle a) Spacefilling representation of nucleosome core (different colors for 4 different histones: H2A, H2B, H3, H4, 2 copies of each in nucleosome = histone "octamer") b) and c) top and side views of crystal structure of nucleosome with 146 bp of bound DNA Protein = gray, surface contour view DNA = blue, in a left-handed negative solenoidal supercoil that goes 1.8 times around histone complex before going out into "linker" segment for connection to next "bead". Wrapping around the histone core removes about 1 turn, introducing a negative supercoil, which is compensated by adjacent DNA (linker) becoming positively supercoiled to keep Lk constant (with no DNA strand breakage), but adjacent DNA becomes relaxed by the action of a topoisomerase that removes a turn (changes Lk by -1), leaving one net negative supercoil in each nucleosome core particle. Schematic drawing relates (c) to beads on a string schematic drawing above. Other proteins, include histone H1 and a number of nonhistone proteins, also bind to the DNA in chromatin.

 Molecular Biology Techniques