Chapter 2: The Structure of DNA
I. Introduction
A. Introduction: General Reasons For Why DNA Holds Large Amounts of Information
1. In the previous chapter, we learned how it was determined that DNA held the information of heredity
a. Meischer
b. Weismann
c. Avery, MacLeod and McCarty
d. Hershey and Chase
2. What is so special about DNA that gives it the ability to hold important genetic information?
3. The ability for DNA to hold genetic information lies in its structure
4. Compared to proteins, its structure seems quite simple
- DNA has the ability to hold large amounts of stored information
B. Introduction: Types of Nucleic Acids
1. Throughout scientific history, DNA has been called by several different names
a. Nuclein (Meischer)
b. Thymus nucleic acid
c. Nucleic Acid (because it could be found in all cells)
d. Dexoyribonucleic acid (DNA)
2. There are two different types of nucleic acids
a. DNA (Deoxyribonucleic acid)
b. RNA (Ribonucleic acid)
3. Although they actually look chemically similar, these two nucleic acids actually look very different structurally
II. The Building Blocks of DNA
A. DNA Building Blocks: Primary (10) Structure
1. Nucleic acids (whether DNA or RNA) are polymers of repeating subunits known as nucleotides
2. Nucleotides consist of three important components
a. Five carbon sugar (pentoses)
b. A phosphate group
c. A nitrogenous base (can contain many nitrogens)
B. DNA Building Blocks: The Nitrogenous Base Component of DNA
1. The presence of the nitrogenous bases in nucleic acids was discovered by Friedrich Miecher after he started to determine the chemistry of his nuclein
2. They are called nitrogenous bases due to the fact that they are have a high nitrogen content
3. They are considered a base due to the fact that they have the properties of a base (proton acceptors)
4. By and large, when part of the structure of DNA the nitrogenous bases are non-polar, which is important for DNA structure
C. DNA Building Blocks: The Nitrogenous Base Component of DNA
1. There are five different nitrogenous bases that are commonly incorporated into nucleic acids
a. Adenine
b. Guanine
c. Cytosine
d. Thymine
e. Uracil
2. Both DNA and RNA contain Adenine, Guanine, Cytosine
3. DNA contains Thymine
4. Instead of Thymine, RNA has Uracil
5. Adenine and guanine are known as purines and have a double ring
6. Cytosine, Thymine and Uracil are known as pyrimadines and have a single ring
D. DNA Building Blocks: The Nitrogenous Base Component of DNA
1. DNA has a specific composition of nitrogenous bases with respect to one another, which was discovered by the Austrian Biochemist Erwin Chargaff
2. In 1940, Chargaff developed a paper chromatography method to analyze the amount of each nitrogenous base present in a molecule of DNA
3. Chargaff observed several important relationships among the molar concentrations of the different bases
4. In 1940 Chargaff proposed two important rules with regards to the nitrogenous base composition of DNA, which became known as Chargaff’s rules
5. Chargaff rules are as follows
a. [A] = [T]
b. [G] = [C]
c. [A] + [G] = [T] + [C] or the number of purines is equal to the number of pyrimidines
6. Chargaff also found that the base composition, as defined by the percentage of G and C (G+C content) for DNA is the basically the same for organisms of the same species, and different for organisms of different species
7. The G + C content can vary from 22 – 73% depending on the organism
E. DNA Building Blocks: The Pentose Sugars
1. Each nucleotide will contain a pentose (5 carbon) sugar, whether it be a nucleotide that gets incorporated into DNA or RNA
2. The sugar that is used in DNA is deoxyribose, whereas ribose, is used in another type of nucleic acid called RNA
3. Within the ring, there are four carbon atoms (labeled 1’, 2’, 3’ etc) joined by an oxygen atom
4. The fifth carbon (the 5’ carbon) projects upward from the ring
6. Ribose and Deoxyribose differ in structure only by the presence or absence of a 2’ hydroxyl group
a. For RNA, the 2’ carbon has a hydroxyl group bound to it
b. For DNA, the 2’ carbon does not have a hydroxyl group (deoxy) bound, instead it has a hydrogen bound to it
7. The difference in whether there is a 2’ hydroxyl (as in RNA) or a 2’ hydrogen (as in DNA), gives DNA and RNA different chemical properties due to the fact that the hydroxyl group is more reactive than the hydrogen
a. RNA can fold into a greater array of structures
b. DNA is more stable than RNA; RNA is more prone to degradation
E. DNA Building Blocks: The Phosphate Group
1. The phosphate group (PO4) gives DNA and RNA the properties of an acid
2. At physiological pH, the phosphate group is a proton donor
3. The linking bonds that are formed from the phosphates are esters that have the property of being extremely stable, yet are easily broken by enzymatic hydrolysis
F. DNA Building Blocks: Putting Together a Nucleotide
1. Nucleotides are the building blocks of nucleic acids
a. Phosphate group
b. Pentose sugar (Deoxyribose or Ribose)
c. Nitrogenous base (A,C,T,G or U)
2. In a nucleotide, the nitrogenous base is bound to the 1’ carbon of the pentose sugar
3. In a nucleotide the phosphate group is bound to the 5’ carbon
4. A nucleotide can have one, two or three phosphate bound to the 5’ carbon
a. The phosphate that is bound to the 5’ carbon is known as the α phosphate
b. The second phosphate from the 5’ carbon is the β phosphate
c. The third phosphate from the 5’ carbon is the γ phosphate
G. DNA Building Blocks: Nucleosides vs. Nucleotides
1. A nucleoside consists of only a pentose sugar and a nitrogenous base
a. Guanosine (if containing guanine)
b. Cytosine (if containing cytidine)
c. Adenosine (if containing adenine)
d. Thymidine (if containing thymine)
e. Uridine (if containing uracil)
2. A nucleoside becomes a nucleotide once at least one phosphate group is bound to the 5’ carbon)
a. If one phosphate is bound then it is a nucleotide monophosphate (NMP)
b. If two phosphates are bound then the nucleotide is a nucleotide diphosphate
c. If three phophates are bound then the nucleotide is a nucleotide triphosphate
H. DNA Building Blocks: Nucleotide Nomenclature
1. The nucleotide takes its name from the incorporated nitrogenous base, the type of pentose sugar that is used and the number of phosphates present (see previous point)
2. If the nucleotide contains deoxyribose, then it is called a deoxynucleotide
3. If the nucleotide contains ribose, then it is called a ribonucleotide
4. For instance, if the nucleotide contains three phosphates, deoxyribose and guanine it is called deoxyguanosine triphosphate, and is abbreviated dGTP
5. For instance, if the nucleotide contains two phosphates, ribose and adenine, it is called adenosine diphosphate and abbreviated ADP
III. DNA Structure
A. DNA Structure: Polymerization of Nucleotides To Get A Strand of DNA
1. To create a strand of DNA, a polymer of repeating nucleotides must be formed
2. The nucleotides are joined (polymerized) by a condensation reaction
3. The 3’ hyroxyl group on the preceding nucleotide will form an ester bond to the 5’ phosphate of the next nucleotide to be added to the chain
a. In the reaction, a water molecule is lost
b. The bond between the two nucleotides is called a phosphodiester bond, or a 5’ 3’ phosphodiester bond to indicate the polarity of the strand
B. DNA Structure: The Significance of 5’ and 3’
1. Each end of a DNA strand has different chemical properties
2. On one end of the DNA chain, there is a free 5’ PO4
3. On the other end of the DNA chain, there is a free OH group
4. The asymmetry of the ends of a DNA strand gives it a specific polarity
a. The end with the free 5’ PO4 is known as the 5’ end
b. The end with the free 3’ OH is known as the 3’ end
5. The sequence of bases in a single strand of DNA can be written using specific conventions
a. Sequence is always written in the 5’ 3’ direction
b. Sequence of the strand in the figure on the right is GCTA, and when written using proper conventions is 5’ –P-GCTA-OH-3’
C. DNA Structure: The Length of DNA
1. RNA can have a range in length from less than one hundred to many thousands of nucleotides (bases)
2. Cellular DNA molecules can be as large as several hundred million nucleotides
3. When measuring the length of a given DNA strand, we are usually measuring the number of nucleotides, or “bases” present in the DNA strand
4. For double stranded DNA molecules, the number of base pairs (abbreviated bp) is used as a measure of length
5. Generally, the unit of length used for a double stranded DNA molecule is a kilobase pair (kb), which is equal to 1000 bp
6. Some double stranded DNA molecules, such as chromosomes, can be quite large and we can express their size in terms of mega-base pairs (Mb), which is equal to 1,000,000 base pairs
IV. DNA Secondary Structure
A. DNA Secondary Structure: Introduction
1. When Watson and Crick deduced the biologically active structure of DNA, they found it to be more complex than just a string of nucleotides polymerized together
2. Watson and Crick deduced that DNA exists with secondary structure in which there are two interwoven strands and thus is double stranded
3. The structure a double stranded DNA molecule takes is in the form of a double helix
B. DNA Secondary Structure: Hydrogen Bonding Between The Bases
1. To obtain a double stranded molecule of DNA, thermodynamically stable hydrogen bonds must form between the nitrogenous bases on the opposite strands
2. Hydrogen bonds provide one of the forces that hold the two strands together
a. One hydrogen bond alone is weak
b. In a double stranded DNA molecule, there are many hydrogen bonds, which when taken collectively are quite strong
c. Many hydrogen bonds give a double stranded DNA molecule strong structural integrity
3. Only certain nitrogenous bases are able to pair together
a. This type of base pairing is known as Watson-Crick base pairing
b. A pairs with T through 2 hydrogen bonds
c. G pairs with C through 3 hydrogen bonds
4. Watson-Crick base pairing fits the observations made by Erwin Chargaff
5. Watson-Crick base pairing allows the 1’ carbons of the two strands to be exactly the same distance apart (1.08 nm) which aids in the stability of DNA
6. Other types of base pairing can occur in DNA, e.g. G-T base pairing, but these are extremely rare
C. DNA Secondary Structure: Hydrogen Bonding Between The Bases
1. To obtain a double stranded molecule of DNA, thermodynamically stable hydrogen bonds must form between the nitrogenous bases on the opposite strands
2. Hydrogen bonds provide one of the forces that hold the two strands together
a. One hydrogen bond alone is weak
b. In a double stranded DNA molecule, there are many hydrogen bonds, which when taken collectively are quite strong
c. Many hydrogen bonds give a double stranded DNA molecule strong structural integrity
3. Only certain nitrogenous bases are able to pair together
a. This type of base pairing is known as Watson-Crick base pairing
b. A pairs with T through 2 hydrogen bonds
c. G pairs with C through 3 hydrogen bonds
4. Watson-Crick base pairing fits the observations made by Erwin Chargaff
5. Watson-Crick base pairing allows the 1’ carbons of the two strands to be exactly the same distance apart (1.08 nm) which aids in the stability of DNA
6. Other types of base pairing can occur in DNA, e.g. G-T base pairing, but these are extremely rare due to the need to keep the two strands exactly the same distance apart
D. DNA Secondary Structure: Base Stacking and the Watson-Crick Double Helix
1. In most models we see, the structure of the DNA looks like a ladder, with the base pairs being the rungs
a. This is not physiologically correct
b. Due to the fact that DNA is in solution in vivo
2. In DNA, base stacking results in significant chemical stability
3. The deoxyribose/phosphate backbone of the DNA is polar and hydrophilic
4. Meanwhile, the nitrogenous bases as non-polar and hydrophobic – practically insoluble in the aqueous environment in the cell
5. In order to exclude water from the interior of the DNA molecule, the paired, relatively flat nitrogenous bases tend to stack on top of one another by means of a helical twist (base stacking) - looks like a rotating stack of coins
E. DNA Secondary Structure: The Backbone of the Watson Crick DNA Double Helix
1.The backbone of each DNA strand consists of alternating deoxyribose and phosphate groups
2. In order to get appropriate base pairing on the interior, Watson and Crick realized that each strand must run anti-parallel
a. One strand running 5’ 3’
b. The other in the opposite direction (3’ 5’)
3. This structure gives DNA several properties which are important for both DNA replication and for transcription (the first step in gene expression)
F. DNA Secondary Structure: Major and Minor Grooves
1. Looking at the exterior of the DNA Molecule the double helix will create two different grooves
a. Major groove
b. Minor groove
2. The grooves are present because the two bonds that attach a base pair to its deoxyribose sugar rings are not directly opposite
3. These grooves can be large enough for proteins to be able to bind DNA
4. Specifically, the major groove plays a significant role in sequence-specific DNA-protein interactions
V. Different Double Helical Structures
A. Different Double Helical Structures: Introduction
1. The DNA double helix can take three different forms depending on the conditions of the solution the DNA is suspended in
a. Some are physiologically relevant
b. Some are not physiologically relevant because the solution conditions are not observed in vivo
2. The three different forms of DNA are:
a. B-DNA
b. A-DNA
c. Z-DNA
B. The structure of B-DNA
1. The B-DNA form is considered the Watson and Crick conformation
2. The B-DNA is the most predominant form in vivo
3. The B-form of DNA is present when the DNA is present in conditions of high humidity (95%) and relatively low salt
4. In B-DNA, the major groove is wide and of moderate depth
5. In B-DNA the minor groove is also of moderate depth, but is narrower
6. For each turn of the helix there will be 10.5 bp/turn
C. The structure of A-DNA
1. The A-DNA form will be present if the water content is decreased and the salt concentration is increased during crystallization
2. The A-form is not thought to be physiologically relevant
3. The A-DNA form takes the shape of a right-handed double helix
4. The A-DNA form is more compact and slightly tilted
a. The bases are tilted with respect to the axis
b. There are 11 bases per turn
5. The major groove is deep and narrow
6. The minor groove is shallow and broad
D. The structure of Z-DNA
1. The Z-form of DNA was discovered by the Alexander Rich Lab in 1979 (MIT)
2. It can only occur under physiological conditions if the molecule contains particular base sequences long stretches of alternating C’s and G’s
3. The presence of Z-DNA in vivo may be important for regulating gene expression
4. The Z-DNA is a left handed double helix, and turns in a counter-clockwise fashion when viewed down its axis
5. The backbone forms a zig-zag structure
6. In Z-DNA there are 12 bp/turn
7. The major groove is shallow, almost to the point of being non-existent
8. The minor groove is deep and narrow
9. Z-DNA was first formed in the laboratory under high-salt conditions, or in the presence of alcohol
10. Z-DNA can be present under normal physiological conditions if methyl groups are added to the Cytosines
VI. Strand Separation and DNA Melting
A. Strand Separation and DNA Melting: Introduction
1.DNA is generally double stranded – however, it can be denatured
a. Replication (in vivo)
b. Transcription (in vivo)
c. To perform PCR (in vitro)
2. DNA is denatured (melted) in vivo by proteins that are specialized to perform this task
3. In vitro, DNA is most commonly denatured by the use of heat
a. At low heat, DNA is double stranded
b. At high heat, DNA melts and becomes single stranded
B. Strand Separation and DNA Denaturation: Denaturation Kinetics
1. In looking at the structure of DNA, the phosphodiester bonds that help form the backbone are quite strong
2. The hydrogen bonds between the nitrogenous base pairs which hold the two strands of DNA together are quite weak
3. Application of heat will cause:
a. The hydrogen bonds to break and the two DNA strands to separate
b. No effect on the strong phosphodiester bonds that form the backbone
c. At the end of denaturation, the DNA is single stranded
5. If one has a solution of double stranded DNA it is possible to follow the progress of the denaturation after heat is applied
6. Normally, we can measure the presence of DNA solution by absorption of UV light at a wavelength (λ) of 260 nm
7. Generally, double stranded DNA absorbs less UV light at a λ = 260 nm than single stranded DNA
a. Base stacking of double-stranded DNA quenches the ability of the DNA to absorb UV light
b. Native double-stranded DNA will absorb about 40% less UV light as compared to the same amount of single stranded DNA
8. As the DNA denatures its absorption of UV light at λ = 260 nm increases, which is a phenomenon known as hyperchromicity
9. The concentration of double-stranded DNA when the A260 = 1.00 is 50 ug/mL
10. The absorbance of a solution has an A260 = 1.37
11. The temperature at which a double-stranded DNA molecule denatures is denoted by (Tm)
12. The melting temperature denotes the point at which 50% of DNA in solution is single-stranded
13. Melting temperature is dependent on the G-C content of the double-stranded DNA
a. G-C base pairs are formed with three hydrogen bonds
b. A-T base pairs are formed with two hydrogen bonds
c. As the number of hydrogen bonds increases the amount of energy to break the base pair also increases
14. The greater the G-C content of the DNA, the higher the melting temperature will be
15. It is possible to estimate melting temperature without experimentation by using the following formula:
16. Tm = 3(G-C base pairs) + 2 (A-T base pairs)
C. Strand Separation and DNA Denaturation: Reversible Renaturation
1.When heated solutions of denatured DNA are cooled the single stranded DNA will renature
a. Single strands will meet their complementary strands to form a new double helix
b. The process is called renaturation or annealing