Chapter 14: DNA: The Genetic

Material
[Link] Nature of Genetic Material:
1.1. Griffith Finds that Bacterial Cells Can Be Transformed: The first indication
towards the transformative nature of bacterial cells came when British microbiologist
Frederick Griffith’s tried to make a vaccine against the influenza, which was thought to to
be caused by a bacteria called Streptococcus pneumoniae. This bacteria is found to exist in
two forms, the S form which causes pneumonia and the R form which does not. The S form
of such a bacteria is the one that exists normally in nature with a virulent polysaccharide
coat, while the R form is a mutant of the S form and lacks the virulent polysaccharide coat.
The S strand is called as such due to it forming smooth colonies on culture dish, while the
R strand is called as such due to it forming rough colonies on culture dish. The mutant R
strand lacks an enzyme found in the S strand responsible for forming the polysaccharide
coat. Griffith’s experiment was done through injecting mice with both strands in hopes of
understanding the difference between them. The results of his experiments were as follows:

1.2. Griffith’s Interpretation: Griffith performed four experiments where various

variables were taken into consideration to detect the difference between both strands.
Griffith came to a conclusion that the polysaccharide coat cannot cause pneumonia alone,
but rather it needs the bacteria to work accordingly. However, in Griffith’s fourth
experiment, a shocking realization came when he injected both a dead S strand and a living
R strand where live S form pneumonia were found within the mice’s lungs. Griffith came
to a conclusion that the polysaccharide coat moved from the dead S strand to the living R
strand. The phenomenon was then called transformation. Transformation is then the
transfer of genetic information from one cell to another.

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 1.3. Avery MacLeod, and McCarty Identify the Transforming Principle: Avery,
MacLeod, and McCarty repeated Griffith’s experiment and made sure to remove all the
protein from the transforming material and achieved a 99.98% purity. Despite protein
removal, the transforming activity of bacteria was not reduced. Their experiments
supported the identity of DNA as the substance transferred between cells by transformation
and indicated that the genetic material, at least in this bacterial species, is DNA. The
deduction came through deriving five properties that resembled those of DNA which are:
1. The elemental composition agreed closely with that of DNA.
2. When spun at high speeds in an ultracentrifuge, it migrated to the same level (density)
as DNA.
3. Extracting lipids and proteins did not reduce transforming activity.
4. Protein-digesting enzymes did not affect transforming activity, nor did RNA-digesting
enzymes.
5. DNA-digesting enzymes destroyed all transforming activity.
1.4. Hershey and Chase Demonstrate that Phage Genetic Material is DNA:
Avery’s results, though factual, were not widely accepted due to many biologists believing
that proteins were the source of genetic material. That was until Hershey and Chase
experimented with viruses that affected bacteria called bacteriophages, commonly known
as phages. Viruses are known to be much simpler than bacteria, as they consist of genetic
material, either DNA or RNA, surrounded by a protein coat. The reason why Hershey and
Chase used bacteriophages is the need for a simple system to differentiate between the
roles of proteins and that of DNA. Through identifying what molecule entered the bacteria,
Chase and Hershey could identify the source of genetic material. Through identifying
unique molecules to DNA and Protein, Chase and Hershey were able to identify which of
DNA or protein were the ones to enter the bacteria. The labeling was done through using
radioactive 32P to label DNA and radioactive 35S for protein. The reason for such
radioactive labeling is the presence of phosphorus in DNA but not in Protein and the
presence of Sulfur in some protein but never in DNA. As such, two experiments were done
by Hershey and Chase. In one, viruses were grown on a medium containing 32P, which was
incorporated into DNA; in the other, viruses were grown on medium containing 35S, which
was incorporated into coat proteins. Each culture of labeled viruses was then used to infect
separate bacterial cultures. After infection, the bacterial cell suspension was agitated in a
blender to remove the infecting viral particles from the surfaces of the bacteria. This step
ensured that only the part of the virus that had been injected into the bacterial cells—that is,
the genetic material—would be detected through centrifugation to produce a pallet for
analysis. In the 32P experiment, a large amount of radioactive phosphorus was found in the
cell pellet, but in the 35S experiment, very little radioactive sulfur was found in the pellet.
Hershey and Chase deduced that the genetic information viruses injected into bacteria was
DNA, and not protein.

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 1.5. Scientific Thinking of Hershey’s and Chase’s Experiment:

2. DNA Structure:
2.1. Discovery of DNA: Frederick Miescher discovered DNA when he extracted a white
substance from the nuclei of humans and fish sperm. This substance was different from all
other substances which led Miescher to believe he discovered a new biological molecule.
He called the biological molecule a nuclein due to its association to the nucleus. He then
named it nucleic acid due to it being slightly acidic in nature.
2.2. DNA Components and 3D Structure: Although a DNA’s structure was yet to be
discovered by Miescher, the DNA’s components were known to be:
1. A 5-carbon sugar
2. A phosphate (PO4) group that is attached to the carbon sugar
3. A nitrogen-containing base (Nitrogenous Base). The base could either be a purine, like
Adenine or Guanine, or a pyrimidine, like Thymine and Cytosine. In the case of RNA,
Uracil replaces Thymine as a pyrimidine.

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 2.3. Numbering Scheme of DNA: In order to unambiguously refer to a carbon atom
within a nucleotide, a convention was made where the carbons in the sugar where
numbered with a prime to distinguish them from atoms in bases. As such, carbons were
numbered from 1´ to 5´ starting with the carbon containing Oxygen. Through such a
numeric scheme, it is inferred that a phosphorus is attached to 5´ carbon, the base is
attached to the 1´ carbon, and a hydroxyl group (-OH) is attached to 3´ carbon.
2.4. Phosphodiester Bonds: A phosphodiester bond is formed when two nucleotide
monomers are linked through dehydration reaction between the 5´ phosphate of one
nucleotide and the 3´ of another. The name phosphodiester is given to such a bond due to
the phosphate molecule being bonded to two ribose sugars through a pair of ester bonds.
Linear strands of DNA or RNA, no matter how long, almost always have a free 5′
phosphate group at one end and a free 3′ hydroxyl group at the other. By convention, the
sequence of bases is usually written in the 5′-to-3′ direction.

2.5. Chargaff’s Rules: Chargaff’s experiments showed that the nucleotide composition of
DNA molecules varied in complex ways, depending on the source of DNA. As such, DNA
was no longer believed to be a simple repeating molecule and that DNA might have
information encoding properties that genetic material require. Chargaff’s observation led to
a realization of the base pair complementary rule, where the the amount of adenine present
in DNA always equals the amount of thymine, and the amount of guanine always equals the
amount of cytosine. Chargaff’s observation gave rise to two important findings:
1. The proportion of A always equals that of T, and the proportion of G always equals
that of C, or A = T, and G = C.
2. The ratio of G–C to A–T varies with different species.
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 2.6. Rosalind Franklin’s Discovery of X-Ray Diffraction Patterns of DNA: X-
Ray diffraction is a process where crystals of certain molecules are bombarded with beams
of X-Ray. The rays are bent, or diffracted, by the molecules they encounter, and the
diffraction pattern is recorded on photographic film. The patterns resemble the ripples
created by tossing a rock into a smooth lake. When analyzed mathematically, the
diffraction pattern can yield information about the three-dimensional structure of a
molecule. However, gathering DNA crystals seemed to be a problem until British
researcher Maurice Wilkins learned how to prepare uniformly oriented DNA fibers, and
with graduate student Ray Gosling succeeded in obtaining the first crude diffraction
information on natural DNA in 1950. Rosalind Franklin proceeded to enhance the
technique until she discovered that DNA is helical with a diameter of 2 nm and a a
complete turn every 3.4 nm.

2.7. The Watson-Crick Model: Prior to Franklin publishing her results, James Watson
and Francis Crick worked out a likely structure for DNA based on the evidence brought by
previous scientists. It is to be noted that Watson and Crick did not perform a single
experiment themselves related to DNA structure; rather, they built detailed molecular
models based on the information available from Chargaff and Rosalind. Their
understanding of the DNA molecule led them to propose a double helix structure.
2.8. Structure of a Single Strand of DNA: Each DNA strand is made up of repeating
sugar molecules joined by a phosphodiester bond. This joining makes the phosphodiester
backbone of the DNA [Link] helix is often compared to a spiral staircase, in which
the two strands of the double helix are the handrails on the staircase. Each DNA strand
extends from a 5´ to a 3´ direction.

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 2.9. Diagram of a Single Strand of DNA:

[Link] Double Helix Structure: Two DNA strands arrange as a double helix to form
two grooves, a major groove and a minor groove. The double helix arounds around one
axis with a width of 2 nm, perform a complete turn every 3.4 nm, and the distance between
bases is 0.34 nm which are antiparallel.

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 [Link]-Pairing Structure: Adenine is known to form two hydrogen bonds with
Thymine and Guanine is able to form three hydrogen bonds with Cytosine. This
configuration also pairs a two-ringed purine with a single-ringed pyrimidine in each case,
so that the diameter of each base-pair is the same. This consistent diameter is indicated by
the X-ray diffraction data.

3. Basic Characteristics of DNA Replication:

3.1. Meselson and Stahl Demonstrate the Semiconservative Mechanism: There
exists three possible models of DNA Replication which are:
1. The Conservative Model: both strands of the parental duplex would remain intact
(conserved), and new DNA copies would consist of all-new molecules. Both daughter
strands would contain all-new molecules.
2. The SemiConservative Model: One strand of the parental duplex remains intact
in daughter strands (semi-conserved); a new complementary strand is built for each
parental strand consisting of new molecules. Daughter strands would consist of one
parental strand and one newly synthesized strand.
3. The Dispersive Model: copies of DNA would consist of mixtures of parental and
newly synthesized strands; that is, the new DNA would be dispersed throughout each
strand of both daughter molecules after replication.

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 3.2. The Meselson-Stahl Experiment: In order to truly evaluate the correct mechanism
of replication, Meselson and Stahl labeled a DNA and then followed the labeled DNA
through two rounds of replication. The labeling mechanism was done though using the
isotope 15N, which is noticeably denser than its isotope 14N, and then separating the
molecules through ultracentrifugation. After several years in a medium rich in 15N, the 15N
became incorporated into the DNA of bacteria and then transferred to a medium containing
14N, which is lighter than its isotope. At various time intervals, DNA was extracted from

the cells at various times.

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 3.3. Meselson and Stahl’s Results: Meselson and Stahl’s experimental data were used to
provide a basis of validation or nullification of the possible models for DNA replication
which are:
1. The Conservative Model: It was not consistent with the data because after one
round of replication, two densities should have been observed: DNA strands would
either be all-heavy (parental) or all-light (daughter). This model is rejected.
2. The Semiconservative Model: It is consistent with all observations: After one
round of replication, a single density would be predicted because all DNA molecules
would have a light strand and a heavy strand. After two rounds of replication, half of the
molecules would have two light strands, and half would have a light strand and a heavy
strand, and so two densities would be observed. Therefore, the results support the
semiconservative model.
3. The Dispersive Model: It was consistent with the data from the first round of
replication, because in this model, every DNA helix would consist of strands that are
mixtures of 1/2 light (new) and 1/2 heavy (old) molecules. But after two rounds of
replication, the dispersive model would still yield only a single density; DNA strands
would be composed of 3/4 light and 1/4 heavy molecules. Instead, two densities were
observed. Therefore, this model is also rejected.
3.4. DNA Replication Requires a Template, Nucleotide, and a Polymerase
Enzyme: In order for DNA replication to take place, there is a need for something to
copy (Parental DNA molecule), something to do the copying (Enzymes), and building
blocks to make copies (Nucleoside Triphosphate). DNA replication is done through three
major steps: initiation, elongation, and termination. Initiation occurs when replication
begins. Elongation occurs when new strands of DNA are synthesized by DNA polymerase.
Termination occurs when replication is terminated.

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 5. DNA Polymerase: Enzymes that add to the 3´ end of existing strands a new base. DNA
polymerase synthesize in a 5′ to 3′ direction by extending a strand base-paired to the
template. DNA polymerases also require a primer to begin synthesis; they cannot begin
without a strand of RNA or DNA base-paired to the template. RNA polymerases do not
have this requirement, so they usually synthesize the primers.

4. Prokaryotic Replication:
4.1. The E. coli Model: In order to understand a detailed picture of prokaryotic DNA
replication, scientists used the E. Coli as a model to distinguish prokaryotic replication
from eukaryotic replication.
4.2. Prokaryotic Replication Starts at a Single Origin: The first DNA polymerase to
be isolated in the E. Coli was DNA polymerase I (Pol I) which was thought to be the sole
contributor towards DNA replication. That was until a strand was taken lacking Pol I which
still could perform DNA replication. As such, two additional polymerase enzymes were
discovered in E. Coli called DNA polymerase II (pol II) and DNA polymerase III (Pol III).
It was also discovered that some polymerase was able to remove nucleotides rather than
just add them, and some were able to act as nucleases. These nucleases were then
categorized to be either endonuclease or exonuclease. An endonuclease is an enzyme that
cuts DNA internally, while an exonuclease is an enzyme which removes nucleotides from
the end of DNA. Pol I, Pol II, and Pol III have an exonuclease activity from 3´ to 5´
responsible to proof read wrongly paired bases. Pol I also has an exonuclease activity from
5´ to 3´ responsible to remove RNA primers. The three different polymerases have different
roles in the replication process. DNA Pol III is the main replication enzyme; it is
responsible for the bulk of DNA synthesis. DNA Pol I acts on the lagging strand to remove
primers and replace them with DNA. The Pol II enzyme does not appear to play a role in
replication but is involved in DNA repair processes. It is to be noted that these polymerase
are not the only existent ones in E. Coli, but rather there exists up to five discovered
polymerase enzymes.
4.3. Replication is Bidirectional from a Unique Origin: As most prokaryotes, E.
coli possess a circular DNA where the replication of such DNA takes place from a certain
origin. Two separate replisomes are loaded onto the origin and initiate synthesis in the
opposite directions on the chromosome and move from a unique origin to the unique
terminus. These two replisomes continue in opposite directions until they come to a unique
termination site. It is to be noted that any DNA controlled by an origin is known to be a
replicon.
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 [Link] of an E. coli Undergoing DNA Replication:

[Link] DNA Requires Energy and Causes Torsional Strain: One

function of DNA polymerase is unwinding DNA during synthesis; however, DNA
replication is more efficient when the DNA is unwound prior to the polymerase. As such, a
need for an enzyme with a DNA unwinding activity arises. Such an enzyme is known to be
a helicase. Helicase requires energy in the form of ATP, and can progressively unwind
DNA, forming single strands. It is to be noted that such strands are not stable due to them
possessing hydrophobic bases that become exposed to water. As such, another protein is
needed called the single-stranded-binding proteins (SSBs) in order to coat such single
strands. When these strands are separated, a phenomenon occurs to DNA called
supercoiling where the already twisted DNA will further coil in space. Supercoiling is
described to be the topological state of DNA. Some enzymes act to relieve the topological
state of DNA and are generally known to be topoisomerase. The topoisomerase involved in
DNA replication is called DNA Gyrase.

[Link] is Semidiscontinuous: DNA is known to be an antiparallel structure

where one strand moves from the 5´ to the 3´ direction while the other moves from the 3´ to
5´. As such, there lies some constraints posed on DNA replication due to the nature of DNA
and the direction by which the DNA polymerase can synthesize a strand, from the 5´ to the
3´ direction. This implicates that polymerase synthesizes DNA in opposite directions. It
should also be noted that DNA polymerase requires primers which needs to be added once
the helix opens up. This means that one strand can be synthesized in a continuous fashion
from an initial primer, but the other strand must be synthesized in a discontinuous fashion
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 with multiple priming events and short sections of DNA being assembled. The strand that is
continuous is known as the leading strand, while the strand that is discontinuous is called
the lagging strand. It is to be noted that the DNA fragments found on the lagging strand are
called Okazaki fragments, after the man who discovered discontinuous synthesis.

[Link] Occurs at the Replication Fork: The partial opening of a DNA helix to
form two single strands has a forked appearance, and is thus called the replication fork.
Enzymatic activities are known to occur at the replication fork. However, it should be noted
that the synthesis on the leading strand and the lagging strand differs from one another in a
manner where:
1. Priming: The enzyme DNA primase synthesizes the primers required by the DNA
polymerase during replication. This enzyme is an RNA polymerase that synthesizes
short stretches of RNA 10 to 20 base-pairs long that function as primers for DNA
polymerase. The RNA primer is then removed and replaced with DNA.
2. Leading-Strand Synthesis: Synthesis on the leading strand is done through a
single priming event and extended indefinitely by the action of DNA Pol III. When the
enzymes remains attached to a template, it can synthesize around the entire circular E.
Coli chromosome. This ability is known to be called processivity. Pol III has high
processivity due to the existence of β subunits. The β subunit is made up of two
identical protein chains that come together to form a circle. This circle can be loaded
onto the template like a clamp to hold the Pol III enzyme to the DNA The β subunit
forms a “sliding clamp” to hold the Pol III enzyme to the DNA. For the clamp to
function, it must be opened and then closed around the DNA through a clamp loader.

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 [Link] of a DNA polymerase Sliding Clamp:

3. Lagging-Strand Synthesis: Due to the discontinuous nature of the lagging

synthesis, various steps are needed to replicate this strand. Primase is needed to
synthesize primers for each Okazaki fragment, and then all these RNA primers must be
removed and replaced with DNA. Finally, the fragments need to be stitched together.
The Okazaki fragments themselves are synthesized by DNA Pol III, like the leading
strand, but the primers are removed and replaced with DNA by DNA Pol I. Pol I then
removes all primers and replaces them with DNA. Once this happens, the nick between
Okazaki fragments after primer removal is sealed by DNA ligase.

4. Termination: Termination occurs on a site of the DNA that is roughly the opposite of
the origin of replication in circular chromosomes. The last stages of replication produce
two daughter molecules that are intertwined like two rings in a chain. These intertwined
molecules are unlinked by the same enzyme that relieves torsional strain at the
replication fork: DNA gyrase.

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 [Link]: Replisomes are macro-molecules formed by enzymes involved in DNA
replication. The replisome has two main subcomponents: the primosome, and a complex of
two DNA Pol III enzymes, one for each strand. The primosome consists of primase and
helicase, along with a number of accessory proteins. The two Pol III complexes include two
synthetic core subunits, each with its own β sliding clamp subunit.
4.9. Diagram of the Replication Fork:

5. Eukaryotic Replication:
5.1. The Complexity of Eukaryotic Replication: The complexity of Eukaryotic
replication exists due to the larger amounts of DNA organized in multiple chromosomes,
and the linear structure chromosomes possess. This complexity is also quite apparent in the
enzymatic activity required for dealing with the end of chromosomes and packaging.
5.2. Eukaryotic Replication Uses Multiple Origins: The number of DNA and how it
is packed in linear chromosomes poses a problem in eukaryotes as they possess multiple
large chromosomes that each is larger than that of the E. coli chromosome. If each
chromosome had a single origin, the time needed for replication would be quite high.
Eukaryotic cells reduce the time needed for replication by using multiple origins for each
chromosome, resulting in multiple replicons. These origins are not sequence-specific, and
their recognition depends on chromatin structure and sequence both. It is to be noted that
the number of origins could be adjusted.
5.3. Complexity of Eukaryotic Fork: Prior to the S-phase, of interphase, Helicase
complexes are loaded onto potential replication origins but not activated. As S-phase starts,
a subset of these complexes are activated and the rest of replisomes are assembled. These

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 replisome differ from the ones found in Prokaryotic Cells in a manner such that the priming
process is done through a complex of DNA polymerase α and primase. Such a complex is
responsible for synthesizing primers composed of RNA and short stretches of DNA. It is to
be noted that each of the leading and lagging strand has its own polymerase complex. DNA
polymerase epsilon (Pol ε) is responsible for leading strand synthesis and DNA polymerase
delta (Pol δ) does the lagging strand.
5.4. The Clamping Subunit in Eukaryotes: In Eukaryotes, the sliding clamp subunit
that allows the enzyme complex to stay attached to the template is called PCNA (for
proliferating cell nuclear antigen). The PCNA sliding clamp forms a trimer, but has similar
structure and function to the β subunit sliding clamp.
5.5. Archaean and Eukaryotic Replication Proteins are Evolutionary Related:
For DNA polymerases, they are differentiated based on non-homologous regions into seven
subfamilies. The subfamily used by eukaryotes and archaea is the same, but different from
the one used by bacteria. As for replicative helicase complexes, all have a similar structure,
forming a ring around a single strand of DNA, but the bacterial and archaeal/eukaryal
enzymes are not homologous, and even move in the opposite direction along DNA. Lastly,
the primase component of the replisome is also not homologous between bacteria and
archaea/eukarya. The one function that is clearly homologous in all domains of life is the
processivity factors: sliding clamps and clamp loaders.
5.6. Linear Chromosomes Have Specialized Ends: At the end of eukaryotic
chromosomes, there exists specialized ends called telomeres. These structures protect the
ends of chromosomes from nucleases and maintain the integrity of linear chromosomes.
Telomeres are also composed of specific DNA sequences, but they are not made by the
replication complex.
5.7. Replicating Ends: The very structure of a linear chromosome causes a cell problems in
replicating the ends. The directionality of polymerases, combined with their requirement
for a primer, creates this problem. The leading strand can be completely replicated, but the
lagging strand cannot be finished. When the last primer is removed, it cannot be replaced.
During the next round of replication, when this shortened template is replicated, it will
produce a shorter chromosome.

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 5.8. Action of Telomerase: Telomeres are composed of short repeated sequences of DNA
and synthesized by an enzyme called telomerase, which is known to use an internal RNA
as a template rather than the DNA itself. Telomerase contains an internal RNA that the
enzyme uses as a template to extend the DNA of the chromosome end. Multiple rounds of
synthesis by telomerase produce repeated sequences. This single strand is completed by
normal synthesis using it as a template

5.9. Regulation of Telomerase: Telomerase activity is developmentally dependent and

regulated where during childhood, telomerase activity is perceived to be high, but low in
most somatic adult cells. The reason why adult somatic cells have low activity of
telomerase is through preventing the expression of the gene that encodes the telomerase
enzyme. There also exists a relation between telomerase and and cell senescence (aging).
Support for the relationship between senescence and telomere length comes from
experiments in which telomerase was introduced into fibroblasts in culture. These cells had
their lifespan increased relative to controls that had no added telomerase. Furthermore,
cancer cells do continue to divide indefinitely, and this would not be possible if their
chromosomes were being continually shortened. Cancer cells generally show activation of
telomerase, which allows them to maintain telomere length; but this is clearly only one
aspect of conditions that allow them to escape normal growth controls.

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6. DNA Repair:
6.1. DNA Damage Constantly Occurs: Not only does errors occur in DNA replication,
but they also occur when cells are exposed to DNA-damaging-agents. These DNA
damaging agents could be UV rays, chemicals in the environment, or X-rays. These
damaging agents could potentially cause genetic mutation or increase the rate of mutation
from background levels and become mutagens. It should be noted that mutagens could also
be detected in diets, either from contaminated or natural plants.
6.2. DNA Repair Restores Damaged DNA: DNA repair is a process to restore damaged
DNA to its native state. It is to be noted that there exists multiple pathways for DNA repair
and reversing damage for each cell. Such systems assist in reducing mutational load on
organisms to reach an acceptable range. DNA repair falls into two general categories:
specific and nonspecific. Specific repair systems target a single kind of lesion in DNA and
repair only that damage. Nonspecific forms of repair use a single mechanism to repair
multiple kinds of lesions in DNA.
6.3. Specific Systems of Repair: DNA repair could be done through a series of systems
depending on the conditions and how DNA was damaged/mutated. Some of these systems
are:
1. Mismatch Repair: For proofreading DNA polymerase, DNA mutations occur in a
lower rate than predicted due to the action of Mismatch Repair (MMR). MMR removes
incorrect bases which were added and replaces them with the correct base through
copying from the template DNA. For mismatch repair to occur, there is a need for the
system to be able to distinguish between the template strand and the newly synthesized
strand. In E. Coli, the system recognizes the template strand from the synthesized
strand through the methylation of Adenine in the sequence 5´GATC 3´. This sequence
is a palindrome, so the complement (3′ CTAG 5′) is the same 5′ to 3′, and the A in each
strand is methylated. The methylase responsible does not act immediately after
replication, leaving a short window for the MMR system to be able to identify the
newly synthesized strand.
2. PhotoRepair: When DNA is exposed to leaking UV rays, adjacent thymine in DNA
become covalently bonded. This process is known to be called a thymine dimer. The
need for repair is done through a specific repair pathway, called photorepair, that
recognizes the thymine dimer and reverts it back to normal. The enzyme photolyase
binds to thymine DNA with adjacent thymines dimers, then uses energy from visible
light to cleave the dimer and restore the thymine bases
3. Excision Repair: A common form of nonspecific repair is excision repair. In this
pathway, a damaged region is removed, or excised, and is then replaced by DNA
synthesis. Excision repair follows three steps: recognition of damage, removal of the
damaged region, and resynthesis using the information on the undamaged strand as a
template.

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 6.4. Diagram of the Thymine Dimer being Photorepaired:

6.5. Diagram of a Non-Specific Excision Repair:

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7. General Notes to Take Into Consideration:
1. Bacteriophages: When a phage infects a bacterial cell, it first binds to the cell’s outer
surface and then injects its genetic information into the cell. Once inside the cell, the viral
genetic information uses the cell’s information-processing machinery to make viral
proteins, lead- ing to the production of thousands of new viruses. The buildup of viruses
eventually causes the cell to burst, or lyse, releasing progeny phage.
2. Primer: A primer is a short nucleic acid sequence that provides a starting point for DNA
synthesis. In living organisms, primers are short strands of RNA. The primers are removed
before DNA replication is complete, and the gaps in the sequence are filled in with DNA by
DNA polymerases.
3. Nucleases: A nuclease is an enzyme capable of cleaving the phosphodiester bonds
between nucleotides of nucleic acids. Nucleases variously affect single and double stranded
breaks in their target molecules. In living organisms, they are essential machinery for many
aspects of DNA repair.
4. Replisome: The replisome is a large protein complex that carries out DNA replication,
starting at the replication origin. It contains several enzymatic activities, such as helicase,
primase and DNA polymerase and creates a replication fork to duplicate both the leading
and lagging strand.
5. Clamp Loader: In all cells and in some viruses, the clamp is a ring-shaped protein
complex that encircles DNA, forming a sliding platform on which DNA polymerases and
other proteins that move along DNA are assembled.
6. DNA Ligase: DNA ligase is a DNA-joining enzyme. If two pieces of DNA have
matching ends, ligase can link them to form a single, unbroken molecule of DNA. In DNA
cloning, restriction enzymes and DNA ligase are used to insert genes and other pieces of
DNA into plasmids.
7. Difference Between Synthesis on Lagging Strand between Prokaryotes
and Eukaryotes: Synthesis on the lagging strand differs most from related events in
prokaryotic DNA replication. The Okazaki fragments are significantly shorter: 200 bp
instead of 1,000 bp. As each fragment reaches the previous fragment, synthesis continues to
displace the strand containing the previous primer. The displaced primer is removed by a
nuclease, and the remaining fragments joined by ligase.
8. Mutagen: A mutagen is a chemical or physical phenomenon, such as ionizing radiation,
that promotes errors in DNA replication. Exposure to a mutagen can produce DNA
mutations that cause or contribute to diseases such as cancer.
9. Palindrome Sequence: A palindromic sequence is a nucleic acid sequence in a double-
stranded DNA or RNA molecule whereby reading in a certain direction on one strand is
identical to the sequence in the same direction on the complementary strand.

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