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0 DNA
Deoxyribonucleic acid (DNA) is the genetic material for all prokaryotes, eukaryotes, and many
viruses. DNA is a polymer of nucleotides that forms the genome of an organism, and the genetic
information contained within the genome is encoded in the sequence of the nucleotide bases in the
polymer. The differences in genetic makeup between organisms lies in the length and the nucleotide
sequence of their DNA.
2.11.1 Purines
The purines are nitrogenous bases that contain two fused hetrocyclic rings formed from carbon and
nitrogen. The two purines found in nucleic acids are adenine and guanine, and they form base pairs
with the thymine and cytosine respectively. The structure of adenine is shown below, and the
numbers identify positions on the hetrocyclic rings.
2.11.2 Pyrimidines
The pyrimidines are nitrogenous bases that contain one hetrocyclic ring formed from carbon and
nitrogen. The two pyrimidines found in DNA are thymine and cytosine, and they form base pairs
with the adenine and guanine respectively. Uracil is a pyrimidine that substitutes for thymine in
RNA to base pair with adenine. The structure of cytosine is shown below, and the numbers identify
positions on the hetrocyclic ring.
2.12 Nucleosides
A nucleoside if formed by adding a sugar to the nitrogenous base. Ribose is the sugar found in
RNA and 2-deoxyribose is the sugar in DNA. Carbon 1 of the appropriate sugar is attached to
nitrogen 9 in purines or nitrogen 1 in pyrimidines to form a nucleoside. The carbon atoms in the
sugar are designated as prime (‘) to avoid confusion with positions on the nucleotide ring.
2.12.1 Ribose
Ribose is the pentose sugar found in RNA. Ribose is linked to a nitrogenous base at position 1 and
to phosphate groups at position 5 to form a complete nucleotide. The structure of ribose is shown
below with the carbons numbered.
2.12.2 2-deoxyribose
The pentose sugar found in DNA is 2-deoxyribose. It differs from ribose by lacking a hydroxyl
group at position 2 on the ring. The 2-deoxyribose is linked to a nitrogenous base at position 1 and
to phosphate groups at position 5 to form a complete nucleotide. The structure of 2-deoxyribose
shown below with the carbons numbered.
2.13 Nucleotides
Liking one or more phosphate groups to the 5’ position of the nucleoside sugar produces a
nucleotide. Mono-, di-, and triphosphate forms of nucleotides are commonly found in cells. The
deoxy nucleotide triphosphates used to make DNA are dATP, dCTP, dTTP, and dGTP. RNA is
made from ATP, GTP, CTP, and UTP. The bonds between the phosphate groups are energy rich
and are hydrolyzed to provide energy for cellular processes.
2.2 DNA Structure
DNA is a polynucleotide consisting of a series of 5’-3’ phosphate-deoxyribose linkages that form a
backbone from which the bases extend from. The 5’-3’ linkages confer directionality to the DNA
strands that are wrapped around each other to form a right handed double helix. The DNA strands
are complimentary to each other and are anti parallel with respect to the sugar-phosphate backbone.
The helix is 20A in diameter and makes a complete turn every 34A. The bases are stacked 3.4A
apart and extend into the interior of the helix. Inside the helix, basepairs are formed with the
complimentary base on the opposite strand and hydrophobic interactions between the stacked bases
stabilize the helix. The hydrophilic surface of the helix is formed by the sugar-phosphate backbone.
The double helix can be denatured by heat, pH changes that cause the bases to ionize, and organic
solvents. While denaturing, the absorbance of the DNA solution at 260 nm will increase as the
DNA becomes single stranded. Because of the additional hydrogen bond between GC basepairs,
GC rich DNA will be more difficult (higher denaturation temperature) to denature than AT rich
DNA. When the denaturing agent is removed, the DNA strands will reanneal to form the double
helix. DNA can exist in several helical forms (A, B, and Z). The B form of DNA is shown below.
2.21 Supercoiling
In living cells, DNA is usually constrained from free rotation by proteins and RNA that closely
associate with the double helix. When forces act on the DNA to twist the molecule around its own
axis, supercoils are produced to relieve the tension. One supercoil is formed for every time the
molecule is twisted about its axis, and if the magnitude of the torsion therefore proportional to the
number of supercoils present. Winding the DNA in a direction opposite the clockwise turns of the
helix produces negative supercoils reflecting the underwinding of the helix, and this reflects the
natural state of DNA in vivo. If the magnitude of the negative supercoiling is great enough,
localized disruption of baseparing can occur. Overwinding the DNA in a clockwise direction will
produce positive supercoils, but this is occurs only during laboratory manipulations of DNA. To
maintain an appropriate magnitude of negative supercoiling in a cell, topoisomerase enzymes
expend ATP to induce or relieve supercoils as necessary. Type 1 topoisomerases break one sugar
phosphate backbone, allow the DNA to rotate freely, and then reattach the backbone. This enzyme
can reduce positive and negative supercoils in an ATP dependent manner. Type 2 topoisomerases
are capable of breaking both sugar-phosphate backbones in the helix to relieve or induce tension as
needed. Type 2 topoisomerases do not require ATP for activity.
2.32.1 Chromatin
Human cells have 23 pairs of chromosomes, representing over 3 billion base pairs with a total DNA
length of approximately 2 meters per cell. To compact the DNA to fit in 5-10 uM cell and to protect
the molecule from shearing, DNA is complexed with protein and organized into chromatin. The
fundamental unit of chromatin is the nucleosome core which consists of a histone octamer
composed of two copies of histone H2A, H2B, H3, and H4. This core particle then serves as a
spool to wind up 146 base pairs of DNA, forming a nucleosome. Histone H1 then binds to the
nucleosome, and a chromatin fiber then forms. These fibers form higher order structures such as
100 and 300 angstrom fibers that form the Laemli loops that are anchored to an acidic protein
scaffold to form a highly compact chromosome. The distinctive light and dark banding patterns
seen when the chromosomes are stained are used to identify the individual chromosomes.
2.32.2 DNA Sequences
The DNA sequences in eukaryotic genomes can be divided into nonrepetitive and repetitive DNA
sequences. Nonrepetitive DNA, such as structural genes, are unique sequences that appear only a
few (<20) times in a genome. Repetitive DNA is often found thousands of times in a genome, and
serves no known purpose but makes up nearly 50% of the genome in mammals. Moderately
repetitive DNA occurs about 100-1000 times per genome, while highly repetitive DNA can be
found 10,000 times per genome.
The genes in eukaryotes can be transcribed by RNA polymerase I, II, or III and have a promoter that
is specific for one polymerase. These genes are often regulated singly and may have upstream and
downstream control elements to give a high degree of transcriptional regulation mediated by an
array of transcription factors. These transcription factors interact with the DNA at specific
sequences such as the TATA box found in RNA polymerase II promoters to allow the polymerase to
bind to the promoter. When transcribed, eukaryotic mRNA contains introns that must be spliced out
before the mRNA can be exported from the nucleus for translation.
2.32.4 Variations in Chromosome Number
Polymerase alpha Replication and Priming 5'-3' Polymerase and 3'-5' Exonuclease
Polymerase beta Repair 5'-3' Polymerase, 3'-5' and 5'-3' Exonuclease
Polymerase gamma Mitochondrial Replication 5'-3' Polymerase, 3'-5' and 5'-3' Exonuclease
Polymerase delta Replication 5'-3' Polymerase and 3'-5' Exonuclease
Nitrous acid is a common chemical agent that oxidatively deaminates cytosine, converting it to
uracil. When replicated, this uracil is replaced by a thymine to complete the conversion of a C-G
basepair to a T-A basepair. Nitrous acid is also capable of deaminating adenine to change an A-T
basepair to a G-C basepair.
2.62 Transversions
Transversions are mutations that effect a single base, converting it from a purine to a pyrimidine or
a pyrimidine to a purine. Transversions can produce silent, missense, and nonsense mutations.
2.63 Insertions and deletions
Insertions and deletions are mutations that insert or remove a stretch of one or more nucleotides
within a DNA sequence. Mobile genetic elements such as transposons or insertion sequences cause
insertions in deletions. Insertions and deletions are noted for their ability to cause frameshift
mutations.
2.64 Photoreactivation
In prokaryotes, pyrimidine dimers are recognized by DNA photolyase. This enzyme binds to the
pyrimidine dimer and breaks the covalent links formed by UV light between the adjacent bases in a
light dependent process. The DNA photolyase is then released from the DNA after directly
repairing the potential mutation in the DNA.
2.65 Excision Repair
In prokaryotes altered basepairs, such as pyrimidine dimers, are recognized by the UvrA/B/C
complex. This complex cleaves the sugar-phosphate backbone 7 nucleotides 5’ to the lesion and 3
nucleotides 3’ to the lesion. This short stretch of DNA is removed by the helicase activity of UvrD
to allow DNA polymerase I to synthesize new DNA to replace the excised section. DNA ligase then
reforms the sugar-phosphate backbone. A similar repair system is found in eukaryotes, and when
defective in humans, it leads to a serious disease called xeroderma pigmentosum.
2.66 AP Repair
When the bases are removed from DNA by ionizing radiation or a DNA glycosylase, an AP
endonuclease cleaves the sugar-phosphate backbone near the apurininc or apyrimidininc site and an
exonuclease removes a short stretch of DNA containing the lesion. The resulting gap is then filled
in by DNA polymerase I and the sugar-phosphate backbone is reformed by DNA ligase.
2.67 Mismatch Repair
The mismatch repair apparatus follows behind replication forks to correct errors in replication.
Because the template strand of DNA is methylated at the A residues in GATC sequences and the
new DNA is not, it is possible to recognize which strand of DNA should be corrected. Mismatches
are recognized by MutS and MutL which bind to the mismatch. The sugar-phosphate backbone of
the new strand of DNA is cleaved on either side of the mismatch by MutH. The helicase activity of
MutU then removes this stretch of DNA so it can be replaced by DNA polymerase I and DNA
ligase.
2.81 Transformation
Transformation is a process where an organism will take up extracellular DNA in a manner that it
could become incorporated into the genome or be maintained as extrachromosomal DNA. Some
cells become receptive to transformation during specific phases of their life cycle, while others will
not be transformable without laboratory manipulation. To make cells competent for
transformation, they can be treated with salts such as CaCl2 or be exposed to high voltages to
produce pores in their membranes. After gaining access to the cell, restriction endonucleases may
attack the DNA. If the DNA survives it can undergo recombination with the genome or be
maintained extrachromosomaly.
2.82 Transduction
Transduction is the process in which prokaryotes, and possibly eukaryotes, can acquire new genetic
material from viruses. If a virus accidentally packages a portion of host genome, it can be carried to
other cells by the virus. When this virus-carried DNA enters a new cell, it can undergo
recombination with the new host’s genome.
2.83 Conjugation
In some prokaryotes, a plasmid containing genes for a pillus confers the ability to transfer genetic
material to other bacteria which do not carry the plasmid. Plasmid containing bacteria, designated
F+, use the pillus to attach to untransformed bacteria (F-) to transfer the plasmid. This produces two
F+ bacteria, and can also result in the transfer of genomic DNA in some cases. Occasionally, the F
plasmid will undergo recombination with the host genome, producing a F’ bacterium. When an F’
bacterium transfers the F plasmid via the pillus, some genomic DNA may be transferred as well.
This transferred genomic DNA could then undergo recombination with the DNA of the new host.
2.84 Bacteriophages
Bacteriophages are viruses that infect bacteria. These viruses can transfer genetic material via
transduction or can alter the genotype of the host with the genetic material of the virus itself.
Because of their ability to carry genetic information bacteriophages are utilized as vectors, in a
manner similar to plasmids. Bacteriophages are studied as model systems for prokaryotic gene
regulation and play an important role in infectious disease. In some cases, such as with toxic shock
syndrome, the infection of a benign bacterium with a phage can make it a virulent pathogen.