DNA Structure and Function September 2, 2024 BIO 14
The history of DNA research began with the discoveries of nucleic acids and the development of the double-helix model. In the 1860's, physician Friedrich Miescher isolated phosphate-rich chemicals (nucleic acids) from white blood cells, which led to the discovery of DNA.
The first bacterial transformation, a process where external DNA is taken up by a cell, changing its morphology and physiology, was reported by British bacteriologist Frederick Griffith.
Griffith's transforming principle was discovered during a 1928 experiment with bacteria and mice. Griffith found that bacteria were capable of bacterial transformation, where bacteria take up or incorporate foreign genetic material from the environment, and the material being transferred was referred to as the transforming principle, later known as DNA, was the carrier of genetic information.
Further research in 1944 by scientists Oswald Avery, Colin MacLeod, and Maclyn McCarty confirmed that DNA was the transforming principle. Two strains of bacteria (R or rough), and (S or smooth) were isolated. After isolating both DNA and RNA by observing that only when DNA was present, the bacteria could be transformed and when DNA was absent or degraded, the transforming stopped.
A 1952 experiment by Martha Chase and Alfred Hershey further concluded evidence of DNA being the carrier of genetic material and not proteins. The experiment involved a bacteriophage, which is a virus that infects bacteria. Hershey and Chase showed that only the DNA of a virus needs to enter a bacterium to infect it. Their experiment provided strong support for the idea that genes are made of DNA. They firmly restated the conclusion that Avery, et al. had more tentatively proposed in 1944.
Chargaff's rules (given by Erwin Chargaff in the late 1940's) state that in the DNA of any species and any organism, the amount of guanine nucleotide should be equal to the amount of cytosine nucleotide and the amount of adenine nucleotide should be equal to the amount of thymine nucleotide. A 1:1 stoichiometric ratio of purine and pyrimidine bases (i.e., A+G=T+C) should exist and this pattern is found in both strands of the DNA.
The 1st Parity Rule: A% = T% and G% = C% The 2nd Parity Rule: that the A% is approximate to T% and the G% is approximate to the C% (1:1 ratio). The Chargaff rules helped develop the double-helix model of DNA by Watson and Crick.
DNA Structure and Sequencing
The building blocks of DNA are nucleotides. The important components of the nucleotide are a nitrogenous (nitrogen-bearing) base, a 5-carbon sugar (pentose), and a phosphate group. The nucleotide is named depending on the nitrogenous base. The nitrogenous base can be a purine such as adenine (A) and guanine (G), or a pyrimidine such as cytosine (C) and thymine (T).
The purines have a double ring structure with a six-membered ring fused to a five-membered ring. Pyrimidines are smaller and have a single six-membered ring structure.
The sugar is deoxyribose in DNA and ribose in RNA. The carbon atoms of the five-carbon sugar are numbered 1', 2', 3', 4', and 5' (1' is read as “one prime”). The phosphate, which makes DNA and RNA acidic, is connected to the 5' carbon of the sugar by the formation of an ester linkage between phosphoric acid and the 5'-OH group (an ester is an acid + an alcohol). In DNA nucleotides, the 3' carbon of the sugar deoxyribose is attached to a hydroxyl (OH) group. In RNA nucleotides, the 2' carbon of the sugar ribose also contains a hydroxyl group. The base is attached to the 1'carbon of the sugar.
The nucleotides combine with each other to produce phosphodiester bonds. The phosphate residue attached to the 5' carbon of the sugar of one nucleotide forms a second ester linkage with the hydroxyl group of the 3' carbon of the sugar of the next nucleotide, thereby forming a 5'-3' phosphodiester bond. In a polynucleotide, one end of the chain has a free 5' phosphate, and the other end has a free 3'-OH. These are called the 5' and 3' ends of the chain. A base attached to a five-carbon sugar represents a nucleoside. A nucleotide is composed of one, two, or three phosphate groups attached to a nucleoside.
Scientists helped determine DNA structure in the 1950's including Frances Crick, James Watson, Linus Pauling, Maurice Wilkins, Rosalind Franklin, which led to the Nobel Prize. Franklin died before the award was given.
Watson and Crick proposed that DNA is made up of two strands that are twisted around each other to form a right-handed helix. Base pairing takes place between a purine and pyrimidine on opposite strands, so that A pairs with T, and G pairs with C (suggested by Chargaff's Rules). Thus, adenine and thymine are complementary base pairs, and cytosine and guanine are also complementary base pairs. The base pairs are stabilized by hydrogen bonds: adenine and thymine form two hydrogen bonds and cytosine and guanine form three hydrogen bonds. The two strands are anti-parallel in nature; that is, the 3' end of one strand faces the 5' end of the other strand.
The sugar and phosphate of the nucleotides form the backbone of the structure, whereas the nitrogenous bases are stacked inside, like the rungs of a ladder. Each base pair is separated from the next base pair by a distance of 0.34 nm, and each turn of the helix measures 3.4 nm. Therefore, 10 base pairs are present per turn of the helix. The diameter of the DNA double-helix is 2 nm, and it is uniform throughout. Only the pairing between a purine and pyrimidine and the antiparallel orientation of the two DNA strands can explain the uniform diameter.
DNA Sequencing Techniques
The techniques used to sequence DNA were once complicated and expensive. Then Fred Sanger developed the sequencing method used for the human genome sequencing project (1990-2003), which is also commonly used today.
Sanger's method was called the dideoxy chain termination method, where the dideoxynucleotides are dye-color labeled by letter (A, T, G, C) and used to generate DNA fragments of different lengths, which can then be read. This method is a targeted technique that determines the order and identity of the four nucleotide bases in a DNA segment.
Gel electrophoresis is a laboratory technique used to separate DNA fragments of different sizes, RNA, or proteins by size and electrical charge using a gel made of the chemical agarose (polysaccharide polymer from seaweed). The technique works by passing an electrical current through a gel with small pores, which allows the molecules to move through.
DNA is packed in prokaryote cells in the nucleoid region on a single, circular chromosome where the DNA is twisted by a supercoiling method with the help of certain proteins and enzymes.
In eukaryote cells, however, chromosomes contain a linear DNA molecule wrapped around proteins called histones that form structures called nucleosomes. Nucleosomes are linked to each other with linker DNA, or "beads on a string structure."
DNA Replication Models include conservative, semi-conservative, and dispersive.
In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together.
The semi-conservative method suggests that each of the two parental DNA strands acts as a template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand.
In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.
DNA Replication in Prokaryote Cells
The replication of DNA is rapid in E.coli bacteria (1,000 nucleotides per second).
A key enzyme in DNA replication is DNA polymerase or DNA pol which adds nucleotides one-by-one to the growing DNA chain that is complementary to the template strand. Energy is required to add nucleotides and this energy comes from nucleoside triphosphates (ATP, GTP, TTP, CTP) and the energy source to drive the polymerization.
Three main polymerases in prokaryotes: DNA pol I, II, and III.
Replication of DNA begins at the particular nucleotide sequence called the origins of replication. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds of the nitrogen base pairs. Replication forks (y-shaped structures) are formed as DNA opens up. Single strand binding proteins coat the single strands of DNA near the replication fork and prevent winding back into the double helix.
Primase is the enzyme that makes the RNA primer, which is needed for DNA pol to start synthesis of a new DNA strand.
Primer is the short stretch of nucleotides that is required to initiate replication; RNA nucleotides. Topoisomerase prevents the over-winding of the DNA double-helix.
During replication, the continuously synthesized strand is called the leading strand. The other strand is extended away from the replication fork in small fragments called Okazaki fragments, each of which requires a primer to start the synthesis. The strand with these fragments is called the lagging strand.
A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides.
RNA primers are replaced with DNA by the exonuclease and the primers are removed by the exonuclease.
The ligase is the enzyme that catalyzes the formation of a phosphodiester linkage between the 3' OH and 5' phosphate ends of the DNA
DNA Replication Steps
1 DNA unwinds at the origin of replication.
2 Helicase opens up the DNA-forming replication forks; these are extended bidirectionally.
3 Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
4 Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
5 Primase synthesizes RNA primers complementary to the DNA strand.
6 DNA polymerase III starts adding nucleotides to the 3'-OH end of the primer.
7 Elongation of both the lagging and the leading strand continues.
8 RNA primers are removed by exonuclease activity.
9 Gaps are filled by DNA pol I by adding dNTPs.
10 The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.
Enzymes and Protein Specific Functions in Prokaryotic DNA Replication
DNA pol I
Removes RNA primer and replaces it with newly synthesized DNA
DNA pol III
Main enzyme that adds nucleotides in the 5'-3' direction
Helicase
Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases
Ligase
Seals the gaps between the Okazaki fragments to create one continuous DNA strand
Primase
Synthesizes RNA primers needed to start replication
Sliding Clamp
Helps to hold the DNA polymerase in place when nucleotides are being added
Topoisomerase
Helps relieve the strain on DNA when unwinding by causing breaks, and then resealing the DNA
Single-strand binding proteins (SSB)
Binds to single-stranded DNA to prevent DNA from rewinding back.
DNA Replication in Eukaryote Cells
Eukaryote genomes are larger and more complex than prokaryote genomes, however, the essential steps in replication are the same as in prokaryotes.
The differences between prokaryotic and eukaryotic DNA replication:
Origin of replication in prokaryotes is single, in eukaryotes it is multiple.
Rate of replication in prokaryotes is 1000 nucleotides/s Rate of replication in eukaryotes: 50 to 100 nucleotides/s
DNA polymerase types in prokaryotes (5) eukaryotes (14)
Telomerase is not present in prokaryotes, but present in eukaryotes
RNA primer removal in Prokaryotes: DNA pol I Eukaryotes: RNase H
Strand elongation in Prokaryotes: DNA pol III Eukaryotes: Pol α, pol δ, pol ε
Sliding clamp in Prokaryotes: Sliding clamp in Eukaryotes: PCNA
Telomeres are the DNA structures located at the end of linear chromosomes (in eukaryotes) that protect them from fraying and tangling. Telomerase is the enzyme that contains a catalytic part and an inbuilt RNA template. The telomerase functions to maintain telomeres at chromosome ends.
DNA Repair Methods
Proofreading is the ability of DNA to polymerase itself where the DNA pol reads the newly added base before adding the next one, so a correction can be made. The polymerase enzyme checks whether the newly added base has paired correctly with the base in the template strand. If an incorrect base is added, it is removed so that a correct one can be added.
Mismatch repair is when errors are corrected not during DNA replication, but after replication is completed. Certain repair enzymes recognize the mis-paired nucleotide and cut out the part of the strand that contains it. The cut out portion is then repaired.
Nucleotide excision repair is similar method to mismatch repair, except that it is used to remove damaged bases rather than mismatched ones. When the damaged segments are removed, it is replaced with the correctly paired nucleotide by the DNA pol and sealed with ligase.
Errors during DNA replication cause mutations to occur. Mutations are variations to the nucleotide sequence of the genome. Mutations can also occur from damaged DNA.
Mutations from damaged DNA are two types: Induced mutations result from exposure to chemicals, UV radiation, X-rays, or poisonous gases. Spontaneous mutations are not the result of the environment, but rather result from natural reactions in the body. Point mutations affect only a single base pair.
Substitution mutations are the most common, where one base is replaced with another. Transition substitution mutations are when a purine is replaced with a purine or a pyrimidine is replaced with another pyrimidine. Transversion substitution mutations are when a purine is replaced by a pyrimidine or a pyrimidine is replaced by a purine.
Mutations can also be increased copies of the same codon. Mutations can cause a base to be added, the removal of a base, or a piece of DNA from one chromosome can be lost to another chromosome or to another portion of the same chromosome. These mutations can cause certain types of cancers.
The history of DNA research began with the discoveries of nucleic acids and the development of the double-helix model. In the 1860's, physician Friedrich Miescher isolated phosphate-rich chemicals (nucleic acids) from white blood cells, which led to the discovery of DNA.
The first bacterial transformation, a process where external DNA is taken up by a cell, changing its morphology and physiology, was reported by British bacteriologist Frederick Griffith.
Griffith's transforming principle was discovered during a 1928 experiment with bacteria and mice. Griffith found that bacteria were capable of bacterial transformation, where bacteria take up or incorporate foreign genetic material from the environment, and the material being transferred was referred to as the transforming principle, later known as DNA, was the carrier of genetic information.
Further research in 1944 by scientists Oswald Avery, Colin MacLeod, and Maclyn McCarty confirmed that DNA was the transforming principle. Two strains of bacteria (R or rough), and (S or smooth) were isolated. After isolating both DNA and RNA by observing that only when DNA was present, the bacteria could be transformed and when DNA was absent or degraded, the transforming stopped.
A 1952 experiment by Martha Chase and Alfred Hershey further concluded evidence of DNA being the carrier of genetic material and not proteins. The experiment involved a bacteriophage, which is a virus that infects bacteria. Hershey and Chase showed that only the DNA of a virus needs to enter a bacterium to infect it. Their experiment provided strong support for the idea that genes are made of DNA. They firmly restated the conclusion that Avery, et al. had more tentatively proposed in 1944.
Chargaff's rules (given by Erwin Chargaff in the late 1940's) state that in the DNA of any species and any organism, the amount of guanine nucleotide should be equal to the amount of cytosine nucleotide and the amount of adenine nucleotide should be equal to the amount of thymine nucleotide. A 1:1 stoichiometric ratio of purine and pyrimidine bases (i.e., A+G=T+C) should exist and this pattern is found in both strands of the DNA.
The 1st Parity Rule: A% = T% and G% = C% The 2nd Parity Rule: that the A% is approximate to T% and the G% is approximate to the C% (1:1 ratio). The Chargaff rules helped develop the double-helix model of DNA by Watson and Crick.
DNA Structure and Sequencing
The building blocks of DNA are nucleotides. The important components of the nucleotide are a nitrogenous (nitrogen-bearing) base, a 5-carbon sugar (pentose), and a phosphate group. The nucleotide is named depending on the nitrogenous base. The nitrogenous base can be a purine such as adenine (A) and guanine (G), or a pyrimidine such as cytosine (C) and thymine (T).
The purines have a double ring structure with a six-membered ring fused to a five-membered ring. Pyrimidines are smaller and have a single six-membered ring structure.
The sugar is deoxyribose in DNA and ribose in RNA. The carbon atoms of the five-carbon sugar are numbered 1', 2', 3', 4', and 5' (1' is read as “one prime”). The phosphate, which makes DNA and RNA acidic, is connected to the 5' carbon of the sugar by the formation of an ester linkage between phosphoric acid and the 5'-OH group (an ester is an acid + an alcohol). In DNA nucleotides, the 3' carbon of the sugar deoxyribose is attached to a hydroxyl (OH) group. In RNA nucleotides, the 2' carbon of the sugar ribose also contains a hydroxyl group. The base is attached to the 1'carbon of the sugar.
The nucleotides combine with each other to produce phosphodiester bonds. The phosphate residue attached to the 5' carbon of the sugar of one nucleotide forms a second ester linkage with the hydroxyl group of the 3' carbon of the sugar of the next nucleotide, thereby forming a 5'-3' phosphodiester bond. In a polynucleotide, one end of the chain has a free 5' phosphate, and the other end has a free 3'-OH. These are called the 5' and 3' ends of the chain. A base attached to a five-carbon sugar represents a nucleoside. A nucleotide is composed of one, two, or three phosphate groups attached to a nucleoside.
Scientists helped determine DNA structure in the 1950's including Frances Crick, James Watson, Linus Pauling, Maurice Wilkins, Rosalind Franklin, which led to the Nobel Prize. Franklin died before the award was given.
Watson and Crick proposed that DNA is made up of two strands that are twisted around each other to form a right-handed helix. Base pairing takes place between a purine and pyrimidine on opposite strands, so that A pairs with T, and G pairs with C (suggested by Chargaff's Rules). Thus, adenine and thymine are complementary base pairs, and cytosine and guanine are also complementary base pairs. The base pairs are stabilized by hydrogen bonds: adenine and thymine form two hydrogen bonds and cytosine and guanine form three hydrogen bonds. The two strands are anti-parallel in nature; that is, the 3' end of one strand faces the 5' end of the other strand.
The sugar and phosphate of the nucleotides form the backbone of the structure, whereas the nitrogenous bases are stacked inside, like the rungs of a ladder. Each base pair is separated from the next base pair by a distance of 0.34 nm, and each turn of the helix measures 3.4 nm. Therefore, 10 base pairs are present per turn of the helix. The diameter of the DNA double-helix is 2 nm, and it is uniform throughout. Only the pairing between a purine and pyrimidine and the antiparallel orientation of the two DNA strands can explain the uniform diameter.
DNA Sequencing Techniques
The techniques used to sequence DNA were once complicated and expensive. Then Fred Sanger developed the sequencing method used for the human genome sequencing project (1990-2003), which is also commonly used today.
Sanger's method was called the dideoxy chain termination method, where the dideoxynucleotides are dye-color labeled by letter (A, T, G, C) and used to generate DNA fragments of different lengths, which can then be read. This method is a targeted technique that determines the order and identity of the four nucleotide bases in a DNA segment.
Gel electrophoresis is a laboratory technique used to separate DNA fragments of different sizes, RNA, or proteins by size and electrical charge using a gel made of the chemical agarose (polysaccharide polymer from seaweed). The technique works by passing an electrical current through a gel with small pores, which allows the molecules to move through.
DNA is packed in prokaryote cells in the nucleoid region on a single, circular chromosome where the DNA is twisted by a supercoiling method with the help of certain proteins and enzymes.
In eukaryote cells, however, chromosomes contain a linear DNA molecule wrapped around proteins called histones that form structures called nucleosomes. Nucleosomes are linked to each other with linker DNA, or "beads on a string structure."
DNA Replication Models include conservative, semi-conservative, and dispersive.
In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together.
The semi-conservative method suggests that each of the two parental DNA strands acts as a template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand.
In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.
DNA Replication in Prokaryote Cells
The replication of DNA is rapid in E.coli bacteria (1,000 nucleotides per second).
A key enzyme in DNA replication is DNA polymerase or DNA pol which adds nucleotides one-by-one to the growing DNA chain that is complementary to the template strand. Energy is required to add nucleotides and this energy comes from nucleoside triphosphates (ATP, GTP, TTP, CTP) and the energy source to drive the polymerization.
Three main polymerases in prokaryotes: DNA pol I, II, and III.
Replication of DNA begins at the particular nucleotide sequence called the origins of replication. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds of the nitrogen base pairs. Replication forks (y-shaped structures) are formed as DNA opens up. Single strand binding proteins coat the single strands of DNA near the replication fork and prevent winding back into the double helix.
Primase is the enzyme that makes the RNA primer, which is needed for DNA pol to start synthesis of a new DNA strand.
Primer is the short stretch of nucleotides that is required to initiate replication; RNA nucleotides. Topoisomerase prevents the over-winding of the DNA double-helix.
During replication, the continuously synthesized strand is called the leading strand. The other strand is extended away from the replication fork in small fragments called Okazaki fragments, each of which requires a primer to start the synthesis. The strand with these fragments is called the lagging strand.
A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides.
RNA primers are replaced with DNA by the exonuclease and the primers are removed by the exonuclease.
The ligase is the enzyme that catalyzes the formation of a phosphodiester linkage between the 3' OH and 5' phosphate ends of the DNA
DNA Replication Steps
1 DNA unwinds at the origin of replication.
2 Helicase opens up the DNA-forming replication forks; these are extended bidirectionally.
3 Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
4 Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
5 Primase synthesizes RNA primers complementary to the DNA strand.
6 DNA polymerase III starts adding nucleotides to the 3'-OH end of the primer.
7 Elongation of both the lagging and the leading strand continues.
8 RNA primers are removed by exonuclease activity.
9 Gaps are filled by DNA pol I by adding dNTPs.
10 The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.
Enzymes and Protein Specific Functions in Prokaryotic DNA Replication
DNA pol I
Removes RNA primer and replaces it with newly synthesized DNA
DNA pol III
Main enzyme that adds nucleotides in the 5'-3' direction
Helicase
Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases
Ligase
Seals the gaps between the Okazaki fragments to create one continuous DNA strand
Primase
Synthesizes RNA primers needed to start replication
Sliding Clamp
Helps to hold the DNA polymerase in place when nucleotides are being added
Topoisomerase
Helps relieve the strain on DNA when unwinding by causing breaks, and then resealing the DNA
Single-strand binding proteins (SSB)
Binds to single-stranded DNA to prevent DNA from rewinding back.
DNA Replication in Eukaryote Cells
Eukaryote genomes are larger and more complex than prokaryote genomes, however, the essential steps in replication are the same as in prokaryotes.
The differences between prokaryotic and eukaryotic DNA replication:
Origin of replication in prokaryotes is single, in eukaryotes it is multiple.
Rate of replication in prokaryotes is 1000 nucleotides/s Rate of replication in eukaryotes: 50 to 100 nucleotides/s
DNA polymerase types in prokaryotes (5) eukaryotes (14)
Telomerase is not present in prokaryotes, but present in eukaryotes
RNA primer removal in Prokaryotes: DNA pol I Eukaryotes: RNase H
Strand elongation in Prokaryotes: DNA pol III Eukaryotes: Pol α, pol δ, pol ε
Sliding clamp in Prokaryotes: Sliding clamp in Eukaryotes: PCNA
Telomeres are the DNA structures located at the end of linear chromosomes (in eukaryotes) that protect them from fraying and tangling. Telomerase is the enzyme that contains a catalytic part and an inbuilt RNA template. The telomerase functions to maintain telomeres at chromosome ends.
DNA Repair Methods
Proofreading is the ability of DNA to polymerase itself where the DNA pol reads the newly added base before adding the next one, so a correction can be made. The polymerase enzyme checks whether the newly added base has paired correctly with the base in the template strand. If an incorrect base is added, it is removed so that a correct one can be added.
Mismatch repair is when errors are corrected not during DNA replication, but after replication is completed. Certain repair enzymes recognize the mis-paired nucleotide and cut out the part of the strand that contains it. The cut out portion is then repaired.
Nucleotide excision repair is similar method to mismatch repair, except that it is used to remove damaged bases rather than mismatched ones. When the damaged segments are removed, it is replaced with the correctly paired nucleotide by the DNA pol and sealed with ligase.
Errors during DNA replication cause mutations to occur. Mutations are variations to the nucleotide sequence of the genome. Mutations can also occur from damaged DNA.
Mutations from damaged DNA are two types: Induced mutations result from exposure to chemicals, UV radiation, X-rays, or poisonous gases. Spontaneous mutations are not the result of the environment, but rather result from natural reactions in the body. Point mutations affect only a single base pair.
Substitution mutations are the most common, where one base is replaced with another. Transition substitution mutations are when a purine is replaced with a purine or a pyrimidine is replaced with another pyrimidine. Transversion substitution mutations are when a purine is replaced by a pyrimidine or a pyrimidine is replaced by a purine.
Mutations can also be increased copies of the same codon. Mutations can cause a base to be added, the removal of a base, or a piece of DNA from one chromosome can be lost to another chromosome or to another portion of the same chromosome. These mutations can cause certain types of cancers.