Unit 4 DNA Replication
Key Unit Competence
To be able to explain the process of DNA replication and its significance to livingorganisms.
LEARNING OBJECTIVES
At the end of this unit, the learner will be able to:
• Determine how the structure of DNA enables it to reproduce itself accurately.
• Appreciate the importance of proper DNA replication.
• State semiconservative replication as a process by which DNA unzips and each new molecule
of DNA (daughter DNA) contains one intact strand from the original DNA (parent DNA)
and one newly synthesised strand.
• Apply knowledge of complementary base pairing in DNA to interpret Meselson and Stahl’s
experiment to test different hypothetical models for DNA replication using E.coli grown in a
heavy nitrogen (15N) medium.
• Acknowledge improper DNA replication would result into genetic changes in the nucleus
that would have both positive and negative effects on organisms. For example, changes in
the metabolism of cells, variation that can result into evolution and mutations that may lead
to death.
• State the role of enzymes involved in replication of DNA.
• List the ingredients used to make DNA in a test tube.
• Describe how semi-conservative replication of DNA takes place.
• State that conservative and dispersive replications are other hypothesis for DNA replication.• Explain the importance of DNA replication in organisms.
DNA replication is the process by which DNA makes a copy of itself during cell division.
It produces two identical replicas from one original DNA molecule. This biological processoccurs in all living organisms and is the basis for biological inheritance.
INTRODUCTORY ACTIVITY
Mr Aaron is a senior teacher of Biology. He said: “ I used to illustrate the process of
DNA replication from the diagram below and my students could interpret the process withconfidence. However, a question often came back to me …”
1. Make research from internet or library to explain how this diagram can serve to
explain the process of protein synthesis
2. Mr Aaron said a question often came back from students. On your side, what
challenge you face by using such a diagram?
4.1 MODELS OF DNA REPLICATION
ACTIVITY 4.1
Look in books and on the internet how DNA replicates. Research on semi-conservative model
of DNA replication. Also, try to find other models of DNA replication and present yourfindings to class.
4.1.1 Semi-conservative Model
In 1953, Watson and Crick proposed their classic paper postulating a double helix for DNA.
A month later, they published another paper suggesting how such base-paired structures in DNA
might duplicate itself. The essence of Watson and Crick suggestion is that if DNA molecule
was untwisted and the two strands get separated, each strand could act as a template for
the synthesis of a new complementary strand of DNA. And this new complementary strand
could then be bound to the parental strand of DNA. This model replication is known as the
semiconservative model. It is because half of the parent strand of DNA is retained by newly
formed daughter DNA strand (Figure 4.1).
4.1.2 Other Two Models of DNA Replication
Apart from semiconservative replication model, two other models for DNA replication werealso proposed at that time. The two other DNA replication models are:
Conservative DNA Replication Model
In this model, the two parental DNA strands come together right after replication; and as a
whole, these two parental DNA strands serve as template for the synthesis of completely new
daughter DNA strands. As a result, one daughter DNA molecule contains parental DNA strands,
while the other daughter DNA molecule contains newly synthesized DNA strands (Figure 4.1).
Dispersive DNA Replication Model
In this model, the parental double helix is broken or cleaved into double-stranded DNA that acts
as templates for the synthesis of new double helix molecules. The segments then reassemble into
complete DNA double helices, each with parental and daughter DNA segments interspersed.
After the replication, although the two daughter DNA molecules are identical in their basepair
sequence, the parental double stranded DNA has become dispersed throughout both inthe daughter DNA molecules (Figure 4.1).
4.2 IMPORTANCE OF DNA REPLICATION
Genes duplicate themselves very accurately by DNA replication. The three main importance
of DNA replication are:
Reproduction—One of the most fundamental properties of all living things is the ability to
reproduce. It is through reproduction that parents faithfully pass on their genetic information
specifying their structure and function to their young ones. At organism level, organisms
reproduce either by asexual or sexual reproduction methods. At cellular level, cells duplicate
by cellular division. And at the genetic level, the genetic material duplicates by DNA replication.
Repair—DNA is the centre of instructions that govern the cell’s protein production, growth,
and many other activities in the cells. With this enormity of precise responsibility, any minor
mistakes in the replication process can bring potentially harmful changes in the cell’s behaviour or
for that matter, the whole organism. Therefore, DNA employs various error repair mechanisms
to ensure accurate DNA replication.
Growth—DNA Replication is required for the growth of organisms. DNA replication occurs
in two different forms of cellular division. They are mitosis and meiosis. In mitosis, a single
parent cell divides and gives rise to two identical daughter cells. Each of the daughter cells
has the exact amount of genetic material. For example – Growth of limbs, organs, hair etc.
On the other hand, in meiosis, cells divide and give rise to two haploid sex cells. Thus, DNAreplication plays a vital role in both mitosis and meiosis.
APPLICATION 4.1
1. State the three hypotheses that support the mechanism of DNA replication
2. What is the importance of DNA replication?
4.3 EXPERIMENTAL EVIDENCE OF SEMI-CONSERVATIVE DNAREPLICATION
ACTIVITY 4.3
If semi-conservative model is the correct model of DNA replication, research and present
your case by supporting it with experimental evidence. Also tell how semi-conservative modeldisproved other models of replication.
In 1958, Matthew Meselson and Franklin Stahl performed the experiment to test and prove
that DNA replication is semi-conservative. In their experiment, they used two isotopic forms
of nitrogen, 14N (light) and 15N (heavy), to distinguish newly synthesized strands of DNA from
old strands.
Initially, Meselson and Stahl grew E.coli (bacteria) for many generations in a medium containing
15N-labelled ammonium chloride (15NH4Cl) to incorporate this heavy isotope of nitrogen into
their DNA molecule. As expected, the DNA strands in the bacteria had 15N-15N (heavy) DNA(Figure 4.2).
In the second stage, they transferred the 15N-labelled bacteria to a medium containing nitrogen in
the normal 14N form (light). Then the bacteria were allowed to reproduce for several generations.
Since, the bacteria were grown in the normal 14N form, the entire newly synthesized DNA after
the transfer was now labelled with 14N.
Samples of E.coli were taken at various time periods as the bacteria continued to reproduce
in the medium. The DNAs from these bacteria were extracted and analysed to determine its
density. They determined the density of extracted DNAs by using equilibrium density gradient
centrifugation technique. This technique uses Cesium Chloride (CsCl), a heavy metal salt
that forms solutions of very high density. Thus, they analysed the extracted DNA by simplymixing it with a solution of cesium chloride and then centrifuged at high speed.
As a density gradient of cesium chloride is established by the centrifugal force, the DNA
molecules float “up” and sink “down” within the gradient to reach their equilibrium density
positions. The difference in density between the heavy (15N) DNA and the light (14N) DNA
causes DNA molecules to rest at different positions by forming bands in the gradient
(Figure 4.2).Final Observations
First Generation (After One Replication Cycle)
When the observation was made after one replication cycle in the 14N medium, the entire DNA
had a density that was exactly intermediate between that of 15N-15N DNA and that of 14N-14N
DNA. The intermediate composition was 15N-14N DNA.
Second Generation (After Two Replication Cycles)
Again when the observation was made after two replication cycles, half of the DNA was that
of intermediate density (15N-14N DNA) and half was that of the density of 14N-14N DNA.
The observations (Figure 4.2) made in this experiment exactly tested and proved the predication
of the semi-conservative model. Therefore, through this experiment it has been known that
DNA replication follows semi-conservative model. At the same time, it disproved the claimthat DNA replication follows either conservative or dispersed replication models.
APPLICATION 4.2
1) Complete the following sentences by correct missing words:
(a) .................. is the process by which DNA makes a copy of itself.
(b) ................ and ................. proposed the semi-conservative model of DNA replication.
(c) .................. and .................. are two other models of DNA replication.
2) Why In their experiment, Matthew Meselson and Franklin Stahl used N-15 (e.g. 15NH4Cl)Instead of N-14 alone?
4.4 ENZYMES AND PROTEINS INVOLVED IN DNA REPLICATION
ACTIVITY 4.4
Different parts of a car function together to smoothly and efficiently run a car. DNA replication
is also like a running car. It requires different components or parts to smoothly and efficiently
carry out DNA replication. Now, find out and discuss the enzymes and proteins involved inDNA replication. Make a table suggesting their roles and importance.
4.4.1 DNA Polymerase
In 1955, Arthur Kornberg and his colleagues were the first ones to identify an enzyme that
could synthesize DNA. Back then this enzyme was originally called Kornberg enzyme. But
now it is called DNA polymerase I. And the term DNA polymerase by definition encompasses
enzymes that catalyses the synthesis of DNA.
There are five DNA polymerases.
1. DNA polymerase I
2. DNA polymerase II
3. DNA polymerase III
4. DNA polymerase IV5. DNA polymerase V
4.4.2 DNA Helicase
DNA helicase is an enzyme that unwinds or unzips the double stranded DNA by breaking thehydrogen bonds between the complementary bases.
The action of DNA helicase can be compared with a zipper. When we open a zip, the zipper
runs on a zip and makes a Y-shape structure with the two strands of interlocking teeth. In the
same way, DNA helicase unzips the double stranded DNA and form a Y-shaped fork knownas a replication fork.
4.4.3 Single-strand DNA-binding Proteins (SSB)
In DNA replication when helicase unwinds the double stranded DNA, the two separating
strands of DNA have the tendency to reform or reanneal into double stranded DNA. A protein
called single-strand DNA-binding (SSB) proteins bind to each single-strand DNA and stabilise
them, so that the separating two strands of DNA do not reform double stranded DNA by
complementary base pairing (Figure 4.3).
4.4.4 DNA Ligase
At the end of DNA replication right after the DNA Pol I is removed and replaced all the RNA
primer nucleotides with DNA nucleotides, normally as single-strand nick (gap) is left between
the two DNA fragments (Figure 4.4). This nick is the point where the sugar-phosphate backbone
between adjacent nucleotides is unconnected. So, what DNA ligase does is to join the two
fragments resulting into a longer and continuous DNA strand.
Chemically, DNA ligase catalyses the formation of a phosphodiester bond between the 3′-OHand the 5′-phosphate groups on either side of a nick. As a result, it seals the nick (gap).
Figure 4.4: A flow diagram showing DNA ligase sealing the gap in a new DNA strand
APPLICATION 4.3
1) State the role of each of the following enzymes and proteins in the replication of DNA.
a) DNA Polymerase
b) Primase
c) DNA Helicase
d) Topoisomerase
e) Single-strand DNA-binding Proteins (SSB)
f) DNA Ligase2) Why In their experiment, Matt
4.5 MECHANISM OF DNA REPLICATION
ACTIVITY 4
Scientists have managed to carry out DNA replication in vitro.
They also realized that there is a slight difference between the DNA replication in eukaryotic
cells and in Prokaryotic cells.
Conduct research from available resource and answer the following:
1) What are the requirements to carry out DNA replication in vitro?2) What is the difference between the DNA replication in Prokaryotes and Eukaryotes?
List of the Ingredients used to Make DNA in A Test Tube
Having studied in the vitro DNA synthesis reaction in detail, the scientists have found out
that in order to synthesize DNA, the following components are required for making DNA in
a test tube.
1. A DNA template: The template (original) DNA that is to be copied. A template is a
molecule which is used to make a complementary DNA molecule in the DNA synthesis.
Normally, the two parental DNA strands act as DNA templates.
2. dNTPs (deoxynucleotides): A mixture of four deoxyribonucleoside 5’-triphosphate
(dNTPs) precursors namely: dATP, dGTP, dTTP, and dCTP. These dNTPs are the
precursors for the nucleotide formation in DNA. Without these dNTPs, new DNA cannot
be synthesized.
3. DNA polymerase I (DNA Pol I): An enzyme to carry out DNA synthesis.
4. A primer: A primer is a short DNA sequence that will bind with the single parent DNA
strand and start the DNA synthesis reaction. Without primer, the coming nucleotides
cannot directly base-pair with parent DNA template.
5. Magnesium ions (Mg2+): It is required for DNA polymerase activity to run optimally.
6. Buffer: Provides a suitable chemical environment for optimum activity and stability ofthe DNA polymerase.
4.5.1 Mechanism of DNA Replication in Prokaryotes
Prokaryotic cells are quite simple in structure. They have no nucleus, no organelles and a small
amount of DNA in the form of a single, circular chromosome. Example is Escherichia coli
(E.coli). The mechanism of DNA Replication can be discussed clearly in the following points.
1. DNA replication starts when DNA helicase unwinds or unzips the DNA at the origin
of replication in both the directions.
2. The locally denatured segment of DNA is called the replication bubble. The two separated
parent DNA strands are called template strands.
3. A Y-shaped structure which is formed when DNA unwinds or unzips to expose the
two parent single template strands for DNA replication is called as a replication fork
(Figure 4.5). In most of the cases, replication forks are formed in both sides and, thus,
move simultaneously in two opposite directions. In other words, the movement of
replication fork is bidirectional (Figure 4.5).
4. In the next step, DNA helicase recruits DNA primase enzyme. Primase enzyme synthesizes
a short RNA primer (about 5-10 nucleotides) on the two template strands through whichnew nucleotides are added by DNA polymerase III.
5. The denaturing or separating two strands of DNA have the tendency to reform (reanneal)
double stranded DNA. A protein called single-strand DNA-binding (SSB) proteins
bind to each single-strand DNA and stabilise them, so that the separating two strandsof DNA do not reform double stranded DNA.
6. As the two single-strands of DNA are held in Y-shaped position and are stabilized by
SSB proteins, the DNA polymerase III now comes and starts adding nucleotides by
forming phosphodiester bonds at the 3′-OH of the primer.
7. The DNA polymerase III can add nucleotides only in 5′-3′ directions. However,
the two strands of DNA run in opposite directions forming a polarity. Thus, to
maintain the 5′-3′ polarity of DNA synthesis on both of the two single templates,
DNA is simultaneously made in opposite directions of the two template strands
(Figure 4.6).
(a) The new DNA strand that is synthesized in the same movement of the replication
fork is called the leading strand. This strand requires a single primer for the complete
DNA replication.
(b) On the contrary, the other new DNA strand that is synthesized in the opposite
direction of the movement of the replication fork is called the lagging strand. This
strand requires primers again and again. Therefore, the newly synthesized DNA
strand is discontinuous in nature. And the newly synthesized fragments of DNAon lagging-strand are called Okazaki fragments.
8. The unwinding of the DNA to form a replication fork creates a tension which is relaxed
by DNA gyrase or topoisomerase.
9. At the end of the DNA replication, the RNA primers on the previous Okazaki fragments
are removed by DNA polymerase I.
10. After the DNA polymerase I left, a single-stranded nick is left at the site of the
removal of primer. DNA ligase seals the nick. This completes the process of DNAreplication.
4.5.2 Mechanism of DNA Replication in Eukaryotes
Eukaryotic cells have nucleus, multiple organelles and more DNA arranged in multiple,
linear chromosomes. Examples–Yeast (Saccharomyces cerevisiae), Humans. DNA replication in
eukaryotes (having linear chromosomes) is initiated at multiple sites of origin of replication
(Figure 4.7). In yeast cells, replicators are approximately 100 bp sequence called autonomously
replicating sequences (ARS).
The origin of replication or replicator is located at the centre of each replicon. Replicator is
a DNA sequence that directs the initiation of replication. The initiation of DNA synthesis at
the replicator takes place by a mechanism involving several groups of initiator proteins.
• Firstly, origin recognition complex (ORC), a multi-subunit protein complex, binds to a
replication origin.
• Secondly, mini-chromosome maintenance (MCM) proteins bind to the replication origin.
MCM proteins include several DNA helicases that unwind the double helix.
• Thirdly, helicase loaders, a third set of proteins, mediate binding of MCM proteins to originof origin recognition complex (ORC).
Pre-replication Complex
The complete group of DNA-bound protein is now called as a pre-replication complex. Andat this stage, the DNA is said to be “licensed” for replication.
Replication Bubble (Multiple)
Initiation of DNA synthesis at the origin of replication is followed by formation of replication
bubble. This bubble is formed by two replication forks that begin to synthesize DNA in opposite
directions away from the origin (Figure 4.7). And these bubbles grow in size as replication proceeds
in both 5’ and 3’ end directions. Wherever the growing replication bubble of one replicon meets
the replication bubble of an adjacent replicon, the DNA synthesized by the two replicons is joined
together. Eventually, the DNA synthesized at numerous replication sites is linked together to formtwo double stranded daughter molecules
Significance of Multiple Replicons
In humans, the haploid genome has 24 chromosomes. These chromosomes consist of about
3 billion base pairs long. And eukaryotic chromosome is 25 times longer than the prokaryotic
chromosome. Moreover, the movement of replication fork is much slower in eukaryotes than
in prokaryotes. In this kind of condition, if eukaryotic chromosome has only one origin of
replication or replicator per chromosome, replication of each chromosome would take many
days. So, the question is how does the eukaryotic chromosome replicate faster despite havinga huge amount of chromosomes?
The answer lies in two main characters of the eukaryotic chromosomes. And they are:
(a) DNA replication is initiated at many origins of replication throughout the genome.
(b) DNA replication is bidirectional in nature. In other words, the replication forks move intwo directions at a time.
4.5.3 The Rate of Replication in Prokaryotes and Eukaryotes
Prokaryotes
The genome E.coli consists one replicon with a size of 4.6 Mb (million base pairs, the entire
genome size). The rate of each replication fork movement is about 1000 base pair (bp) per
second. With this rate, E.coli takes about 42 minutes to replicate its entire chromosome.
Eukaryotes
The eukaryotic genome consists of multiple replicons. For example, in humans, there are
about 10,000-100,000 replicons for an average of 30-300 kb (1000 base pairs). And the rate of
replication fork movement is about 100 bp per second. Thus, it takes about 8 hours to replicatethe entire genome.
APPLICATION 4.4
1. Complete the sentence with correct word:
(a) The full form of ORC is ......................... .
(b) The tension ahead of the replication fork is relaxed by ......................... .
(c) The newly synthesized strand is ......................... in nature.
(d) It takes about ......................... hours to replicate the entire genome in Eukaryotes.
(e) DNA replication in Eukaryotes is ......................... in nature.
2. Amoeba and Vibrio cholera are both unicellular human endoparasites. Giving reason,explain which one will undergo rapid DNA replication in vitro.
4.6 MECHANISMS THAT ENSURE ACCURACY OF DNA REPLICATION
ACTIVITY 4.6
It is known that replication is standard. The daughter DNA resemble to the mother DNA.
Carry out a research from available resource to answer the following questions:
1) What factors allow to have accurate DNA replication?2) Discuss what would be outcome of improper DNA replication.
(A) Complimentary Base Pairing
The nitrogen bases of DNA follow the Chargaff ’s rule of base pairing. In simple words, this rule
says that Adenine (A) base pairs with Thymine (T); Guanine (G) base pairs with Cytosine (C).
This base pairing is very strict and accurate. Thus, the complementary base pairing directs theDNA to replicate very accurately and prevents any mistake to occur.
Base pairing between purines and pyrimidines is possible because of hydrogen bonds. We
can simply define hydrogen bond as the attractive force between the hydrogen attached to
an electronegative atom of one molecule and an electronegative atom of a different molecule
(Figure 4.8 b). In the structure of DNA, both the strong electronegative atoms, oxygen (O)
and Nitrogen (N), are partially negatively charged (d–), while the hydrogen (H) has the partial
positive charge (d+). Hydrogen bonds or interactions play very important role in binding thetwo bases of the opposite strands in the DNA.
(B) Semi-conservative Nature of DNA
In DNA replication, two of the original strands of DNA act as templates for new DNA to be
synthesized. So, when the new strands of DNA are synthesized, they are just the complimentary
bases of the two original template strands of DNA. In this way, original sequence of DNA is
semi-conserved with the two original strands of DNA. Thus, the semi-conservative nature ofDNA makes the DNA replication highly accurate.
(C) Proofreading
DNA Pol I and DNA Pol II polymerase enzymes have 3′-to-5′ exonuclease activity, which
means that they can remove incorrectly inserted nucleotides from the 3′ end to 5′ end of the
DNA chain. Thus, they play important role in proofreading mechanism. The insertion of
incorrect nucleotides by both DNA Poly I and DNA Poly III occurs at a frequency of one base
in a million (10–6).
When incorrect nucleotides are inserted in the newly synthesized DNA, the 3′-5′ exonucleases
move backward and remove the incorrect nucleotide from the newly synthesized DNA. Then
they resume the forward movement and insert the correct nucleotides in place of the incorrect
nucleotides. With this proofreading mechanism, the chances of error occurrence in DNAreplication is reduced to one base in a billion (10–9) instead of one base in a million (10–6).
(D) Mismatch Repair
After DNA Replication if there are any mismatched base pairs on the newly synthesized strand,
it can be corrected by methyl-directed mismatch repair. In contrast to proofreading mechanism
where only one base is repaired by DNA polymerase, the mismatch repair mechanism can
replace about thousand bases to make one repair. The Mut family of enzymes plays an importantrole in mismatched repair.
Main Differences Between Prokaryotic and Eukaryotic DNA ReplicationTable 4.1: Differences between prokaryotic and eukaryotic DNA replication
4.7 EFFECTS OF IMPROPER DNA REPLICATION
A. Mutation: A Key to Variability and Evolution
DNA alterations are occasionally beneficial because DNA base-sequence changes, or mutations,
provide the genetic variability that is the raw material of evolution. In other words, mutation
provides variation that can result into beneficial evolution.
Example: Resistance of Bacteria to Drug. The Esherichia coli (bacterium) lives in colon of human
beings. Initially if they are exposed to chloramphenicol, an antibiotic drug, they die. When
Cavilli and Maccacro (1952) exposed the bacteria to high concentration on chloramphenicol,
they found out that the bacteria have mutated and were 250 times resistant to antibiotics. In
this case, mutation has provided variability in the bacterial population to evolve into a new
species which has high survivability against the antibiotic drugs. Thus, mutation is beneficial
for the bacterial population.
B. Lethal Mutation: Mutation that May Lead to Death
One of the best examples is Tay-Sachs disease (TSD). TSD is a fatal autosomal recessive
genetic disorder. It is caused by a mutation in the Hexosaminidase A (alpha polypeptide)
[HEXA gene]. A genetic mutation is a permanent alteration in the DNA sequence that makes up
a gene; this mutation is lethal. It mostly occurs in children and leads to progressive destruction
of the nervous system. When a child with Tay-Sachs reaches the age of three or four years, the
nervous system is severely affected. Eventually, death occurs by the age of five years.
Mutation in Hex-A Gene Causes Tay-Sachs: Tay-Sachs disease results from defects in a
gene on chromosome 15 due to mutation. The gene located on chromosome 15 codes for the
production of the enzyme Hex-A. In normal people, either or both Hex-A genes are active.Thus, the synthesis of this enzyme prevents the abnormal build-up of the GM2 ganlioside lipid.
APPLICATION 4.5
1. Complete the following sentence with correct words:
(a) ............................ is an example of lethal mutation.
(b) ............................... plays an important role in proofreading mechanisms.
(c) Alkaptonuria is also known as ................................... disease.
(d) ...................................... provides variation that can result into beneficial evolution.
(e) Base pairing in DNA occurs due to ................................... bonding.2. Write short not about Tay-Sachs disease (TSD).
4.8 SUMMARY
• DNA replication is the process of producing two identical DNA replicas from one original
DNA molecule.
• DNA replication plays an important role in reproduction, DNA repair and growth of
organisms.
• DNA replicates semi-conservatively, where the two original strands act as template while
the other two strands are newly synthesized.
• The other two models of replication are conservative and dispersive DNA replications.
• Meselson and Franklin Stahl performed the experiment to test and prove that DNA
replication is semi-conservative.
• Enzymes and Proteins Required for DNA Replication are:
— DNA polymerase I and III are functionally required for replication. But DNA
polymerase I, II, IV, V are involved in DNA repair.
— DNA helicase has the role of unzipping or unwinding the double strand structure of
DNA.
— DNA gyrase serves as a main swivel that prevents supercoiling of the DNA ahead
of the replication fork.
— SSB proteins relax the tendency of the two separated DNA strands to reform double
stranded DNA.
— DNA ligase seals the nick at the end.
• The list of the ingredients to make DNA in a test tube involves : a DNA template, dNTPs,
DNA polymerase I, primers, magnesium ion, and buffer solutions.
• In Prokaryotes, DNA replication starts with DNA helicase unwinding or unzipping
the double stranded DNA. Replication forks are formed, then, primers bind on the two
separated strands of DNA. DNA polymerase III starts synthesizing new DNA strands
on both the strands at 5′-3′ directions.
• The synthesis of DNA is discontinuous and thus produces numerous small Okazaki
fragments.
• DNA polymerases I and III have 3′-5′ exonuclease activity. Thus, they remove the primers
and replace the gap with complementary nucleotides.
• In Eukaryotes, replication takes place at multiple sites of origin of replications. In yeast
cells, replicators are approximately 100 bp sequences called autonomously replicating
sequences (ARS).
• Many replicons are formed.
• DNA replication is initiated by multiple proteins.
• Many replication bubbles are formed.
• The main polymerase enzymes involved are: DNA polymerase a, bd, e; they have different
roles.
• The eukaryotic cells have chromosomes 25 times longer than the prokaryotes. Prokaryotes
have only one replicon. Eukaryotes have multiple replicons, which help in replicating
faster than if there were only one replicon.
• The rate of replication in prokaryotes is about 1000 bp per second, whereas the rate of
replication in eukaryotes is about 100 bp per second.
• There are basically four mechanisms that ensure accuracy of DNA replications:
Complimentary base pairing, semiconservative nature of DNA, proofreading, and
mismatched DNA repair.
• Normally uncorrected mistakes in DNA replication are repaired by DNA repair
mechanism. But in very rare cases mistakes are not corrected, leading to mutation.
• On the positive side, mutation can bring species variation and evolution. On the negativeside, mutation can bring defects in metabolic pathway and may also cause death.
4.9 GLOSSARY
• 3′-to-5′ exonuclease activity: DNA Pol I and DNA Pol II polymerase enzymes have
this activity, which means that they can remove incorrectly inserted nucleotides from the
3′ end to 5′ end of the DNA chain.
• Alkaptonuria: It is a rare inherited genetic disorder in which the body cannot process
the amino acids phenylalanine and tyrosine.
• Chargaff ’s rule: This rule says that adenine (A) base pairs with thymine (T);
Guanine (G) base pairs with cytosine (C).
• DNA fyrase: It is an enzyme which serves as a swivel that prevents supercoiling of the
DNA ahead of the replication fork.
• DNA helicase: It is an enzyme which unwinds the double stranded DNA.
• DNA ligase: It is an enzyme which joins the two DNA fragments into a continuous
DNA strand.
• DNA polymerase: It is an enzyme that catalyzes the synthesis of DNA.
• dNTPs: It is a mixture of four deoxyribonucleoside 5′-triphosphate precursors namely:
dATP, dGTP, dTTP, and dCTP.
• Eukaryotes: Eukaryotic cells have nucleus, multiple organelles and more DNA arranged
in multiple, linear chromosomes. Examples: Yeast, Humans.
• Mutation: It is a process by which nucleotide sequence (or base pairs) of DNA is altered.
• Primase: It is an enzyme which synthesizes primer.
• Primer: A primer is a strand of short nucleic acid sequences (generally about 10 base
pairs) that serves as a starting point for DNA synthesis.
• Prokaryotes: They have no nucleus, no organelles and a small amount of DNA in the
form of a single, circular chromosome. Example is Escherichia coli (E.coli).
• Replication fork: It is a Y-shaped structure which is formed when DNA unwinds to
expose the two parent single template strands for DNA replication.
• Semi-conservative model: During DNA replication, each strand of DNA acts as a template
for the synthesis of a new complementary strand of DNA. And this complementary strand
of DNA could then be bound to the parental strand of DNA.
• Single-strand DNA-binding proteins (SSB): These are stabilizing proteins that bind on the
separating two stands of DNA so that they do not reform into double stranded DNA structure.
• Tay-sachs disease (TSD): TSD is a fatal autosomal recessive genetic disorder. It is caused
by a mutation in the Hexosaminidase A (alpha polypeptide) [HEXA gene].
END UNIT ASSESSMENT 4
I. Choose whether the given statements are True (T) or False (F)
1. In conservative DNA replication model, two identical daughter DNA strands are
formed.
2. Meselson and Stahl experiment proved dispersive DNA replication model after two
replication cycles.
3. Without dNTPs, new DNA cannot be synthesized.
4. In Chargaff'’s rule of base pairing. Adenine base pairs with Cytosine.
5. DNA polymerase I has 3′-5′ exonuclease activity.
6. Tay-Sachs disease (TSD) is a fatal autosomal dominant genetic disorder.
7. Topoisomerase II acts as a swivel to prevent supercoiling of DNA.
8. Mutation can be both beneficial and lethal.
9. Synthesis of DNA in lagging strand is discontinuous.10. Primer is required again and again in the leading strand of DNA.
II. Multiple Choice Questions
1. DNA replication plays an important role in
(a) reproduction (b) growth of organisms
(c) DNA repair (d) All of the above
2. Meselson and Stahl are known for
(a) Complementary base pairing
(b) Genetic Code
(c) Semi-conservative DNA replication
(d) Conservative DNA replication
3. DNA helicase has a role to
(a) Seal the nick at the end of DNA replication.
(b) Relax the tendency of DNA to reform double helix structure.
(c) Unwinds or unzips double stranded DNA.
(d) Cut DNA randomly.
4. Which of the following is not an ingredient to make DNA in a test tube?
(a) dNTPs (b) Primers
(c) Magnesium ion (d) Calcium ion
5. The mechanisms that ensure accuracy of DNA replications are
(a) complementary base pairing (b) Proofreading
(c) Mismatched DNA repair (d) All of the above
6. This happens when mistakes are not corrected by DNA repair mechanism.
(a) Mutation (b) Replication
(c) Repair (d) Growth
7. Syntherized fragments of DNA on lagging strand are
(a) Origin complex (b) Mini chromosome
(c) Okazaki fragments (d) Autonomously replicating sequence
8. Mut family protein is important in
(a) Semi-conservative DNA replication
(b) Mismatched repair
(c) Hydrogen bonding
(d) Organizing DNA structure
9. DNA polymerase d appears to perform
(a) Synthesis of DNA on the lagging strand
(b) Synthesis of DNA on the leading strand
(c) Prevention of supercoiling in DNA
(d) Unwinding of DNA
10. The main initiator proteins of DNA replication in eukaryotes consist of three proteins
except
(a) Origin recognition complex
(b) Helicase loaders
(c) Single-stranded binding proteins(d) Minichromosome maintenance proteins
III. Long Answer Type Questions
1. State how complementary base pairing in DNA devises the Meselson and Stahl’s
semiconservative model of replication.
2. Explain different types of DNA replication models.
3. Describe the roles of enzymes involved in DNA replication.
4. Explain the mechanism to show how the structure of DNA enables it to reproduce
itself accurately.
5. Describe the importance of DNA replication in organisms.
6. (i) Identify the structure shown in figure.
(ii) Write the label (a) to (d).
(iii) Describe the process wherein DNA unzips and each new molecule of DNAforms daughter DNA.
7. List out the ingredients used to make DNA in a test tube.
8. Mutation are the ultimate fuel for evolution. Describe mutations as a key genetic
factor in both positive and negative terminology of biology.
Also tell how some people get more sick from bacteria and viruses than others. What
role does our genes play to get that difference?
9. DNA replicates and form a part of HIV-cure-related research. Investigate on thevarious aspects of DNA replication detecting HIV DNA.