Unit-II DNA Replication and Repair

Central Dogma - An Inheritance Mechanism

In molecular biology, central dogma illustrates the flow of genetic
information from DNA to RNA to protein. It is defined as a process in which
the information in DNA is converted into a functional product.
It is suggested that the information present in a DNA is essential to make up
all proteins and RNA acts as a messenger that carries information through
the ribosomes.

Central Dogma Definition

“Central dogma is the process in which the genetic information flows
from DNA to RNA, to make a functional product protein.”

What is Central Dogma?

The central dogma illustrates the flow of genetic information in cells,
the DNA replication, and coding for the RNA through the transcription
process and further RNA codes for the proteins by translation.
The concept of a sequence of interaction can be understood through the
framework. The most common includes biopolymers. The major category of

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biopolymers include Proteins, RNA and DNA that are further divided into
general transfers, unknown transfers, and special transfers.
Special transfers occur in an exceptional case in the laboratory. General
transfer occurs in almost all cells. It describes the regular flow of
information through transcription and translation. Unknown transfers are
said never to occur.

The new DNA strands are formed, with one strand of the parent DNA and
the other is newly synthesized, this process is called semiconservative DNA
replication.

Central Dogma Steps

The central dogma takes place in two different steps:

Transcription
Transcription is the process by which the information is transferred from
one strand of the DNA to RNA by the enzyme RNA Polymerase. The DNA
strand which undergoes this process consists of three parts namely
promoter, structural gene, and a terminator.
The DNA strand that synthesizes the RNA is called the template strand and
the other strand is called the coding strand. The DNA-dependent RNA

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polymerase binds to the promoter and catalyzes the polymerization in the 3′

to 5′ direction.
As it approaches the terminator sequence, it terminates and releases the
newly synthesized RNA strand. The newly released RNA strand further
undergoes post-transcriptional modifications.

Translation
Translation is the process by which the RNA codes for specific proteins. It is
an active process which requires energy. This energy is provided by the
charged tRNA molecules.
Ribosomes initiate the translation process. The ribosomes consist of a larger
subunit and a smaller subunit. The larger subunit, in turn, consists of two
tRNA molecules placed close enough so that peptide bond can be formed at
the expense of enough energy.
The mRNA enters the smaller subunit which is then held by the tRNA
molecules of the complementary codon present in the larger subunit. Thus,
two codons are held by two tRNA molecules placed close to each other and a
peptide bond is formed between them. As this process repeats, long
polypeptide chains of amino acids are synthesized.

Genetic Code

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Unit-II DNA Replication and Repair

Genetic code contains the information of the protein manufactured from

RNA. There are basically three nucleotides and four nitrogenous bases,
which collectively form a triplet codon that codes for one amino acid.
Therefore, the number of possible amino acids range to 4 x 4 x 4 = 64 amino
acids. There are 20 naturally existing amino acids.
The genetic code degenerates. This was explained by the features of the
genetic code, according to which a few amino acids are coded by more than
one codon thus causing them to degenerate. Each codon codes for only one
specific amino acid and the codes are universal irrespective of the type of
organism.
Out of the 64 codons, 3 are stop codons which stop the process of
transcription and one of the codons is an initiator codon i.e. AUG coding for
Methionine.

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Unit-II DNA Replication and Repair

Organization of prokaryotic and eukaryotic chromosomes

The structure and chemical composition of the DNA in both the eukaryotic
and prokaryotic cells are different. The prokaryotic cells have no nucleus, no
organelles and a very small amount of DNA. On the other hand, the
eukaryotic cells have nucleus and cell organelles, and the amount of DNA
present is large.

Prokaryotic DNA vs Eukaryotic DNA

Prokaryotic DNA Eukaryotic DNA

Location

Found freely in the central

Found within the nucleus.
portion of the cytoplasm.

Occurrence

Occurs as a covalent closed Occurs as a linear form of DNA with two

circular form of DNA. ends.

Size

The size of the DNA is less than The size of the DNA is high, usually
0.1 pg in prokaryote. more than 1 pg.

Introns

Introns are absent in the coding Introns are present in the coding region
region of DNA. of DNA.

Nucleosome

There is no formation of
There is a formation of nucleosome.
nucleosome.

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Unit-II DNA Replication and Repair

Quantity of the DNA

The quantity of the DNA is

The quantity of the DNA is more.
comparatively less.

Chromosome

Do not form chromosomes. Forms chromosomes in the nucleus.

DNA Replication

DNA replication occurs in the DNA replication occurs within the

cytoplasm of the cell. nucleus of the cell.

Number of Genes

Prokaryotic DNA contains a Eukaryotic DNA contains a large

small number of gene. number of genes.

Transposons

Prokaryotic DNA lacks Eukaryotic DNA consists of

transposons. transposons.

Number of Chromosomes

Prokaryotic DNA is organized Eukaryotic DNA is organized into many

into a single chromosome. chromosomes.

Histone Proteins

Do not interact with the histone

Associated with the histone proteins.
proteins.

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Difference # Prokaryotic (Bacterial) Chromosome:

1. Structural organization of bacterial chromosome is simple and is
represented mainly by double-stranded DNA molecule. Although there are
specific proteins associated with bacterial chromosome (not the histories)
that help stabilize its supercoiled domains. Compared to eukaryotic
chromosome, one can consider bacterial chromosome to be naked DNA.
2. Bacterial chromosome is covalently closed circular structure consisting of
only a single molecule of DNA (with few exceptions such as Borellia
burgdorfii and Streptomyces the chromosomes of which are linear).
3. Only one bacterial chromosome occurs per bacterial cell (with few
exceptions such as Rhodobacter sphaeroides, a gram-negative phototroph
that possesses 2 chromosomes per cell).
4. Bacterial chromosome contains only a single copy of each gene and is
therefore genetically haploid.
5. Bacterial chromosomes are shorter and contain lesser number of genes.
6. Bacterial chromosome lies free in the cell cytoplasm without any
membrane to separate the chromosome from the cytoplasm., Since the
ribosomes also occur free in cell cytoplasm, the process of transcription and
translation are not spatially separated.
7. Except few, the bacterial DNAs do not contain introns, the noncoding
sequences. As a result, the protein coding genes are not interrupted by
introns and synthesize a single mRNA often containing more than one
coding region. Each coding region independently synthesizes one or more
proteins depending upon the number of operons (Fig. 5.37).

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Unit-II DNA Replication and Repair

Difference # Eukaryotic Chromosome:

1. Structural organization of eukaryotic chromosome is complex as it
contains more than just DNA. In addition to DNA large amount of histone
proteins wound around DNA molecule in a very regular fashion to form
structures called nucleosome. The nucleosomes aggregate to form a fibres
material called chromatin, which itself further compact by folding and
looping to eventually form very dense structure called chromosome.
2. Each eukaryotic chromosome is linear and consists of several pieces of
DNA.
3. Eukaryotic chromosomes are more than one per cell, and this number
varies with the organism. For example, Saccharomyces cerevisiae, a single-
called Baker’s yeast, contains 16 chromosomes arranged in eight pairs,
while human cells contain 46 chromosomes arranged in 23 pairs.
4. Eukaryotic chromosome typically contains 2 copies of each gene and is
therefore genetically diploid. The diploid eukaryotic genome is halved to
haploid via the process called meiosis.
5. Eukaryotic chromosomes are larger and contain greater number of genes.
6. Eukaryotic chromosome occurs in the cell nucleus, which is surrounded
by nuclear membrane that separates chromosome from the cytoplasm while
the ribosomes are in the cytoplasm, the processes of transaction and
translation are spatially separated.

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7. Eukaryotic chromosome contains both introns (noncoding sequence) and

exons (coding sequence). As a result, the protein coding genes are
interrupted by introns. Both introns and exons are transcribed into the
primary RNA transcript from which the nature (functional) mRNA is formed
by excision of introns and transported to the cytoplasm for translation
(protein synthesis) (Fig. 5.38).

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Unit-II DNA Replication and Repair

DNA Replication - Machinery And Enzymes

We know that DNA is self-replicating structure and DNA replicates semi-
conservatively. However, DNA replication is catalyzed by a set of enzymes.
Let’s learn about machinery and enzymes involved in DNA replication.

DNA Replication
In the process of DNA replication, the DNA makes multiple copies of itself. It
is a biological polymerization which proceeds in the sequence of initiation,
elongation, and termination. It is an enzyme-catalysed reaction. DNA
Polymerase is the main enzyme in the replication process.

DNA Replication Process

DNA Replication Steps

Following are the important steps involved in DNA replication:

Initiation
DNA replication demands a high degree of accuracy because even a minute
mistake would result in mutations. Thus, replication cannot initiate
randomly at any point in DNA.
For the replication to begin there is a particular region called the origin of
replication. This is the point where the replication originates. Replication

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Unit-II DNA Replication and Repair

begins with the spotting of this origin followed by the unwinding of the two
DNA strands.
Unzipping of DNA strands in its entire length is unfeasible due to high
energy input. Hence, first, a replication fork is created catalyzed by
polymerases enzyme which is an opening in the DNA strand.

Elongation
As the strands are separated, the polymerase enzymes start synthesizing the
complementary sequence in each of the strands. The parental strands will
act as a template for newly synthesizing daughter strands.
It is to be noted that elongation is unidirectional i.e. DNA is always
polymerized only in the 5′ to 3′ direction. Therefore, in one strand (the
template 3‘→5‘) it is continuous, hence called continuous replication while
on the other strand (the template 5‘→3‘) it is discontinuous replication. They
occur as fragments called Okazaki fragments. The enzyme called DNA ligase
joins them later.

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Unit-II DNA Replication and Repair

Termination
Termination of replication occurs in different ways in different organisms. In
E.coli like organisms, chromosomes are circular. And this happens when the
two replication forks between the two terminals meet each other.

Enzymes Involved In DNA Replication

DNA replication is a highly enzyme-dependent process. There are many
enzymes involved in the DNA replication which includes the enzymes DNA-
dependent DNA polymerase, helicase, ligase, etc. Among them, DNA-
dependent DNA polymerase is the main enzyme.

DNA-dependent DNA polymerase

It helps in the polymerization and catalyzes and regularises the whole
process of DNA replication with the support of other enzymes.
Deoxyribonucleoside triphosphates are the substrate as well as the energy
provider for the replication process. DNA polymerase is of three types:

DNA Polymerase I
It is a DNA repair enzyme. It is involved in three activities:

 5′-3′ polymerase activity

 5′-3′ exonuclease activity
 3′-5′ exonuclease activity

DNA Polymerase II
It is responsible for primer extension and proofreading.

DNA Polymerase III

It is responsible for in vivo DNA replication.

Helicase
Helicase is the enzyme which unzips the DNA strands by breaking the
hydrogen bonds between them. Thus, it helps in the formation of the
replication fork.

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Unit-II DNA Replication and Repair

Ligase
Ligase is the enzyme which glues the discontinuous DNA strands.

Primase
This enzyme helps in the synthesis of RNA primer complementary to the
DNA template strand.

Endonucleases
These produce a single-stranded or a double-stranded cut in a DNA
molecule.

Single-stranded Binding Proteins

It binds to single-stranded DNA and protects it from forming secondary
structures.

DNA Replication in Prokaryotes

The DNA replication in prokaryotes takes place in the following place:

1. The two strands of DNA unwind at the origin of replication.

2. Helicase opens the DNA and replication forks are formed.
3. The DNA is coated by the single-strand binding proteins around the
replication fork to prevent rewinding of DNA.
4. Topoisomerase prevents the supercoiling of DNA.
5. RNA primers are synthesised by primase. These primers are
complementary to the DNA strand.
6. DNA polymerase III starts adding nucleotides at the end of the
primers.
7. The leading and lagging strands continue to elongate.
8. The primers are removed and the gaps are filled with DNA Polymerase
I and sealed by ligase.

DNA Replication in Eukaryotes

The DNA replication in eukaryotes is similar to the DNA replication in
prokaryotes. However, the initiation process is more complex in eukaryotes
than prokaryotes. In eukaryotes, there are multiple origin of replication

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Unit-II DNA Replication and Repair

present. A pre-replication complex is made with other initiator proteins. The

process is entirely the same but the enzymes used are different. E.g. in
eukaryotes, the polymerization process is carried out by the enzyme Pol δ,
whereas in prokaryotes it is done by DNA Pol III.

The Meselson-Stahl experiment

Meselson and Stahl conducted their famous experiments on DNA replication
using E. coli bacteria as a model system.
They began by growing E. coli in medium, or nutrient broth, containing a
"heavy" isotope of nitrogen, ^{15}\text N15Nstart superscript, 15, end
superscript, start text, N, end text. (An isotope is just a version of an
element that differs from other versions by the number of neutrons in its
nucleus.) When grown on medium containing heavy ^{15}\text N15Nstart
superscript, 15, end superscript, start text, N, end text, the bacteria took up
the nitrogen and used it to synthesize new biological molecules, including
DNA.
After many generations growing in the ^{15}\text N15Nstart superscript, 15,
end superscript, start text, N, end text medium, the nitrogenous bases of the
bacteria's DNA were all labeled with heavy ^{15}\text N15Nstart superscript,
15, end superscript, start text, N, end text. Then, the bacteria were switched
to medium containing a "light" ^{14}\text N14Nstart superscript, 14, end
superscript, start text, N, end text isotope and allowed to grow for several
generations. DNA made after the switch would have to be made up
of ^{14}\text N14Nstart superscript, 14, end superscript, start text, N, end
text, as this would have been the only nitrogen available for DNA synthesis.
Meselson and Stahl knew how often E. coli cells divided, so they were able to
collect small samples in each generation and extract and purify the DNA.
They then measured the density of the DNA (and, indirectly, its ^{15}\text
N15Nstart superscript, 15, end superscript, start text, N, end
text and ^{14}\text N14Nstart superscript, 14, end superscript, start text, N,
end text content) using density gradient centrifugation.

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Unit-II DNA Replication and Repair

This method separates molecules such as DNA into bands by spinning them
at high speeds in the presence of another molecule, such as cesium
chloride, that forms a density gradient from the top to the bottom of the
spinning tube. Density gradient centrifugation allows very small
differences—like those between ^{15}\text N15Nstart superscript, 15, end
superscript, start text, N, end text- and ^{14}\text N14Nstart superscript,
14, end superscript, start text, N, end text-labeled DNA—to be detected.

Results of the experiment

When DNA from the first four generations of E. coli was analyzed, it
produced the pattern of bands shown in the figure below:

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Unit-II DNA Replication and Repair

Generation 0
DNA isolated from cells at the start of the experiment (“generation 0,” just
before the switch to ^{14}\text N14Nstart superscript, 14, end superscript,
start text, N, end text medium) produced a single band after centrifugation.
This result made sense because the DNA should have contained only
heavy ^{15}\text N15Nstart superscript, 15, end superscript, start text, N,
end text at that time.

Generation 1
DNA isolated after one generation (one round of DNA replication) also
produced a single band when centrifuged. However, this band was higher,
intermediate in density between the heavy ^{15}\text N15Nstart superscript,
15, end superscript, start text, N, end text DNA and the light ^{14}\text
N14Nstart superscript, 14, end superscript, start text, N, end text DNA.

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The intermediate band told Meselson and Stahl that the DNA molecules
made in the first round of replication was a hybrid of light and heavy DNA.
This result fit with the dispersive and semi-conservative models, but not
with the conservative model.
The conservative model would have predicted two distinct bands in this
generation (a band for the heavy original molecule and a band for the light,
newly made molecule).

Generation 2
Information from the second generation let Meselson and Stahl determine
which of the remaining models (semi-conservative or dispersive) was actually
correct.
When second-generation DNA was centrifuged, it produced two bands. One
was in the same position as the intermediate band from the first generation,
while the second was higher (appeared to be labeled only with ^{14}\text
N14Nstart superscript, 14, end superscript, start text, N, end text).
This result told Meselson and Stahl that the DNA was being replicated semi-
conservatively. The pattern of two distinct bands—one at the position of a
hybrid molecule and one at the position of a light molecule—is just what
we'd expect for semi-conservative replication (as illustrated in the diagram
below). In contrast, in dispersive replication, all the molecules should have
bits of old and new DNA, making it impossible to get a "purely light"
molecule.

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Unit-II DNA Replication and Repair

Generations 3 and 4
In the semi-conservative model, each hybrid DNA molecule from the second
generation would be expected to give rise to a hybrid molecule and a light
molecule in the third generation, while each light DNA molecule would only
yield more light molecules.
Thus, over the third and fourth generations, we'd expect the hybrid band to
become progressively fainter (because it would represent a smaller fraction
of the total DNA) and the light band to become progressively stronger
(because it would represent a larger fraction).

Conclusion
Based on observations and experimental results, Meselson and Stahl
concluded that DNA molecules can replicate semi-conservatively.
Investigation of semi-conservative nature of replication of DNA or the
copying of the cells, DNA didn’t end there. Followed by Meselson and Stahl
experiment, Taylor and colleagues conducted another experiment on Vicia
faba (fava beans) which again proved that replication of DNA is semi-
conservative.

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Unit-II DNA Replication and Repair

Okazaki fragments

An Okazaki fragment is a relatively short fragment of DNA (with an RNA

primer at the 5' terminus) created on the lagging strand during DNA
replication. It was originally discovered in 1968 by Reiji Okazaki, Tsuneko
Okazaki, and their colleagues while studying replication
of bacteriophage DNA in Escherichia coli.

When the lagging strand is being replicated on the original strand, the 5'-3'
pattern must be used; thus a small discontinuity occurs and an Okazaki
Fragment forms. These fragments are processed by the replication
machinery to produce a continuous strand of DNA and hence a complete
daughter DNA helix.

In dealing with the synthesis of complementary DNA strands the

nascent leading strand always reads 5' to 3'. Its antiparallel complement
strand, the nascent lagging strand reads from 3' to 5'. Because the original
strands of DNA are antiparallel, and only one continuous new strand can be
synthesised at the 3' end of the leading strand due to the intrinsic 5'-3'
polarity of DNA polymerases, the other strand must grow discontinuously in
the opposite direction. Regarding the lagging strand, the result of this
strand's discontinuous replication is the production of a series of short
sections of DNA called Okazaki fragments.

Experimental Proof: Each Okazaki fragment is initiated near

the replication fork at an RNA primer created by primase, and extended
by DNA polymerase III. In eukaryotes, lagging strand synthesis is carried out
by the DNA polymerase δ. The primer is later removed by enzymes that have
endonucleolytic activity such as Ribonuclease H (RNAse H), flap
endonucleases (FENs) and Dna2 helicase/nucleases. In prokaryotes the FEN
nuclease is a domain of DNA polymerase I while in eukaryotes FENs are
separate enzymes. The excised RNA bases are replaced with DNA by DNA
polymerase I in prokaryotes or DNA polymerase δ in eukaryotes. Adjoining
fragments are then linked together by DNA ligase, using phosphodiester
bonds, to create a continuous strand of DNA.

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Unit-II DNA Replication and Repair

Okazaki used a pulse chase type experiment to confirm discontinuous

strand replication. He took actively replicating DNA, then added "hot"
tritiated nucleotides for a short pulse of about 5 seconds. During the 5
seconds the radioactive nucleotides were incorporated into the growing DNA
strands. After the pulse Okazaki chased with "cold" un-labeled nucleotides
for varying amounts of time and quickly isolated the DNA. Then the DNA
was centrifuged and analyzed for radioactivity. What Okazaki found was
that with short chases of about 7 to 15 seconds most of the radioactivity
was found in the small fragments higher in the tube after centrifuge.
However with longer chases more radioactivity was found in the lower, larger
strands. This confirmed that during synthesis first small fragments are
formed on the lagging strand, then later these fragments are combined and
incorporated into much larger strands. The small fragments found on the
lagging strand are called Okazaki fragments.

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Unit-II DNA Replication and Repair

Fidelity of DNA replication

Fidelity of replication may depend on a number of factors and also on a
number of steps.
The much higher degree of fidelity actually achieved results largely from the
activities of DNA polymerase. The mechanism by which DNA polymerase
increases the fidelity of replication is by helping to select the correct base for
insertion into newly synthesised DNA.
DNA polymerases are able to discriminate the mismatched base from the
population of correct bases and are able to reject the incorrect base. Such
discrimination power may result from template-induced changes in enzyme
conformation dictating the selection of the correct substrate or increased
binding of the enzyme to the template in the presence of the complementary
nucleotide.
This selectivity appears to increase the accurracy of replication about a
hundredfold and reduces the expected error frequency from 10-4 to
approximately 10~6.
The other major mechanism responsible for the fidelity of DNA replication is
the proofreading activity of DNA polymerases. It has been previously stated
that DNA polymerase I of E.coli has 3′ to 5′ as well as 5′ 3′ exonuclease
activity.
The 5′ to 3′ exonuclease operates in the direction of DNA synthesis and
helps remove RNA primers from Okazaki fragments. The 3′ and 5′
exonuclease acts in the reverse direction of DNA synthesis and participates
in proof-reading of newly synthesised DNA.
Proof-reading is effective because DNA polymerase requires a primer and is
not able to start synthesis de novo. Primers that are hydrogen bonded to the
template are preferentially used when an incorrect base is incorporated, it is
likely to be removed by the 3′ -» 5′ exonuclease activity.
Such 3′ 5′ exonuclease activities are also associated with prokaryotic
polymerase III and eukaryotic polymerases <5 and £. The 3′ ->■ 5′
exonucleases of these polymerases precisely cut the mismatched bases that
have been incorporated at the end of a growing DNA chain, thereby
increasing the accuracy of replication.

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The importance of proof-reading may explain the fact that DNA polymerases
need primers and catalyse the growth of DNA strand only in the 3′ 5′
direction.
When DNA is synthesised in the 5′ -¥ 3′ direction, the energy needed for
polymerisation is obtained from hydrolysis of the 5′ triphosphate group of
free dNTP as it is added to the 3′ hy- droxyl group of the growing chain.
If DNA was to elongated in the 3′ 5′ direction, the energy of polymerisation
would instead have to be come from hydrolysis of 5′ triphosphate group of
the terminal nucleotide already incorporated into DNA. This would eliminate
the possibility of proof-reading.
This is because removal of a mismatched terminal nucleotide would also
eliminate the 5′ triphosphate group required as an energy source for further
chain extension. Therefore, albeit the ability of DNA polymerase to extend a
primer only in the 5′ 3′ direction appears to make replication, it is necessary
for ensuring accurate duplication of DNA.
Therefore, all such error avoidance and post- replicative error correction
mechanisms can increase the fidelity of replication.

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Unit-II DNA Replication and Repair

Overview of differences in prokaryotic and eukaryotic DNA replication

DNA replication in prokaryotes

1. Initiation:

DNA replication begins from origin. In E coli, replication origin is called OriC
which consists of 245 base pair and contains DNA sequences that are highly
conserved among bacterial replication origin. Two types of conserved
sequences are found at OriC, three repeats of 13 bp (GATRCTNTTNTTTT)
and four/five repeats of 9 bp (TTATCCACA) called 13 mer and 9 mer
respectively.

 About 20 molecules of Dna A proteins binds with 9 mer repeats

along with ATP which causes DNA to wraps around dnaA protein
forming initial complex. The dna A protein and ATP trigger the
opening of 13 mer repeats froming open complex.
 Two copies of dnaB proteins (helicase) binds to 13 mer repeats.
This binding is facilitated by another molecule called dnaC. The
dnaB-dnaC interaction causes dnaB ring to open which binds
with each of the DNA strand. The hydrolysis of bound ATP
release dnaC leaving the dnaB bound to the DNA strand.
 The binding of helicase is key step in replication initiation. dnaB
migrates along the single stranded DNA in 5’-3’ direction causing
unwinding of the DNA.
 The activity of helicase causes the topological stress to the
unwinded strand forming supercoiled DNA. This stress is relieved

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Unit-II DNA Replication and Repair

by the DNA topoisomerase (DNA gyrase) by negative supercoiling.

Similarly, single stranded binding protein binds to th separated
strand and prevents reannaeling of separated strand and
stabilize the strand.
 The DNA polymerase cannot initiate DNA replication. So, at first
primase synthesize 10±1 nucleotide (RNA in nature) along the 5’-
3’ direction. In case of E.coli primer synthesized by primase
starts with ppp-AG-nucleotide. Primer is closely associated with
dnaB helicase so that it is positioned to make RNA primer as
ssDNA of lagging strand.

2. Elongation:

 i. Leading strand synthesis:

 Leading strand synthesis is more a straight forward process
which begins with the synthesis of RNA primer by primase at
replication origin.
 DNA polymerase III then adds the nucleotides at 3’end. The
leading strand synthesis then proceed continuously keeping pace
with unwinding of replication fork until it encounter the
termination sequences.

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Unit-II DNA Replication and Repair

 ii. Lagging strand synthesis:

 The lagging strand synthesized in short fragments called Okazaki
fragments. At first RNA primer is synthesized by primase and as
in leading strand DNA polymerase III binds to RNA primer and
adds dNTPS.
 On this level the synthesis of each okazaki fragments seems
straight forward but the reality is quite complex.

Mechanism of Lagging strand synthesis

 The complexicity lies in the co-ordination of leading and lagging

strand synthesis. Both the strand are synthesized by a single
DNA polymerase III dimer which accomplished the looping of
template DNA of lagging strand synthesizing Okazaki fragments.
 Helicase (dnaB) and primase (dnaG) constitute a functional unit
within replication complex called primosome.
 DNA pol III use one set of core sub unit (Core polymerase) to
synthesize leading strand and other set of core sub unit to
synthesize lagging strand.
 In elongation steps, helicasein front of primaseand pol III,
unwind the DNA at the replication fork and travel along lagging
strand template along 5’-3’ direction.

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Unit-II DNA Replication and Repair

 DnaG primase occasionally associated with dnaB helicase

synthesizes short RNA primer. A new B-sliding clamp is then
positioned at the primer by B-clamp loading complex of DNA pol
III.
 When the Okazaki fragments synthesis is completed, the
replication halted and the core sub unit dissociates from their
sliding clamps and associates with new clamp. This initiates the
synthesis of new Okazaki fragments.
 Both leading and lagging strand are synthesized co-ordinately
and simultaneously by a complex protein moving in 5’-3’
direction. In this way both leading and lagging strand can be
replicated at same time by a complex protein that move in same
direction.
 Every so often the lagging strands must dissociates from the
replicosome and reposition itself so that replication can continue.
 Lagging strand synthesis is not completes until the RNA primer
has been removed and the gap between adjacent Okazaki
fragments are sealed. The RNA primer are removed by
exonuclease activity (5’-3’) of DNA pol-I and replaced by DNA.
The gap is then sealed by DNA ligase using NAD as co-factor.

Termination:
 Eventually the two-replication fork of circular E. coli
chromosome meet at termination recognizing sequences (ter).
 The Ter sequence of 23 bp are arranged on the chromosome to
create trap that the replication fork can enter but cannot leave.
Ter sequences function as binding site for TUS protein.

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Unit-II DNA Replication and Repair

 Ter-TUS complex can arrest the replication fork from only one
direction. Ter-TUS complex encounter first with either of the
replication fork and halt it. The other opposing replication fork
halted when it collide with the first one. This seems the Ter-TUS
sequences is not essential for termination but it may prevents
over replication by one fork if other is delayed or halted by a
damage or some obstacle.
 When either of the fork encounter Ter-TUS complex, replication
halted.
 Final few hundred bases of DNA between these large protein
complexes are replicated by not yet known mechanism forming
two interlinked (cataneted) chromosome.
 In E. coli DNA topoisomerase IV (type II) cut the two strand of
one circular DNA and segrate each of the circular DNA and
finally join the strand. The DNA finally transfer to two daughter
cell.

Dr. E. DILIPAN, AP/Biotech Selvam College of Technology

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Unit-II DNA Replication and Repair

DNA replication in Eukaryotes

DNA replication in eukaryotes occur only in S-phase of cell cycle. However,

pre-initiation occur in G1 pahse. Due to sheer size of chromosome in
eukaryotes, chromosome chromosome contains multiple origin of
replication. ARS (autonomously replicating sequence) in case of yeast is
origin for replication.

Steps in DNA replication

1. Initiation
 The first steps is the formation of pre-initiation replication
complex (pre-RC). It occurs in two stage. 1st stage requires, there
is no CDK activities. It occur in early G1 phase. It is known as
licensing but licensed pre-RC cannot initiate replication at G1
phase. 2nd stage is binding of ORC (origin recognition complex).
 The replication begins with binding of ORC to the origin. ORC is
a hexamer of related protein and remains bounded even after
DNA replication occurs. Furthermore ORC is analogue of
prokaryotic dnaA protein.
 After binding of ORC to origin, cdc6/cdc18 and cdtl coordinate
the loading of MEM (mini chromosome maintainance) to origin.
 MEM complex is thought to be major eukaryotic helicase.
 After binding of MEM complex to pre-RC, cdtl get displaced. Then
DdK phosphorylates MEM, which activates its helicase activity.
Again DdK and CdK recruit another protein called cdc45 which
then recruit all the DNA replicating protein such that the origin
get fired and replication begins.

Dr. E. DILIPAN, AP/Biotech Selvam College of Technology

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Unit-II DNA Replication and Repair

2. Elongation:
 DNA polymerase δ synthesizes and adds dNTPs at 3’ end of RNA
primer.
 The leading and lagging strands are synthesized in the similar
fashion as in prokaryotic DNA replication.

Dr. E. DILIPAN, AP/Biotech Selvam College of Technology

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Unit-II DNA Replication and Repair

3. Termination:

 At the end of DNA replication the RNA primer are replaced by

DNA by 5’-3’exonuclease and polymerase activity of DNA
polymerase ε.
 Exonuclease activity of DNA polymerase removes the RNA primer
and polymerase activity adds dNTPs at 3’-OH end preceding the
primer.
 In case of bacteria, with circular genome, the replacement of RNA
primer with DNA is not a problem because there is always a
preceding 3’-OH in a circular DNA.
 But in eukaryotic organism with linear DNA, there is a problem.
When RNA primer at 5’ end of daughter strand is removed, there
is not a preceding 3’-OH such that the DNA polymerase can use
it to replace by DNA. So, at 5’ end of each daughter strand there
is a gap (missing DNA). This missing DNA cause loss of
information contain in that region. This gap must be filled before
next round of replication.

Dr. E. DILIPAN, AP/Biotech Selvam College of Technology

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Unit-II DNA Replication and Repair

 For solving this end replication problem;studies have found that

linear end of DNA called telomere has G:C rich repeats. These
sequence is known as telomere sequence. These repeats of
telomere sequence is different among different organisms.
Telomere in human cell consists of repeats of
TTAGGG/AATCCC. Each species has its own species specific
telomere repeats. These telomere sequence donot codes anything
but it is essential to fill the gap in daughter strand and maintain
the integrity of DNA.

Telomere replication: end replication problem in Eukaryotic DNA

 There is an enzyme found in eukaryotic cell called telomerase.
 Telomerase is a DNA polymerase (RNA dependent DNA
polymerase) which adds many copies of telomere sequence at 3’-
OH end of template strand. Like other DNA polymerase,
terlomerase also adds deoxyribonucleotide at 3’-OH end. Unlike
other DNA polymerase, telomerase adds DNA at 3’-OH end of
parent strand not at the daughter strand and also it synthesizes
the same sequences over and over in absence of template

strand.
 First telomerase binds to 3’-OH end of parent strand by
hybridization between its AACCCCAAC RNA sequences and
TTGGGG DNA sequences (telomere sequences of Tetrahymena).

Dr. E. DILIPAN, AP/Biotech Selvam College of Technology

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Unit-II DNA Replication and Repair

 The telomerase adds TTG at 3’ end of parent strand. After adding

TTG sequences, telomerase translocates along 5’-3’ end of parent
strand. Now the telomerase adds GGGTTG to 3’ end by using its
CCCAAC sequence. Again telomerase translocates and adds
GGGTTA sequence. This process is continued for many time. The
parent strand become more longer than daughter strand. Now
RNA polymerase (PRIMASE) synthesize RNA primer by copying
the parent strand in 5’-3’ direction using telomere sequence as
template.
 The DNA polymerase can now extend the primer in 5’-3’ direction
by adding deoxyribonucleotide to 3’ end.
 The primer is now removed and it won’t be replaced because it is
an extra sequence added by copying telomere sequence.
 Finally the integrity of daughter strand is maintained.
Mechanisms of DNA damage
There are endogenous or exogenous sources of DNA damage, which
may result in mutations and/or cell death if not repaired.
Endogenous sources of DNA damage
 Replication errors
Repetitive sequences: DNA polymerase is error-prone in reading repetitive
DNA sequences. The repetitive region is repeatedly read, especially in triplet
repeats.
Keto-enol tautomerism: a transient tautomeric shift of nucleotide bases
from the usual keto-configuration to the enol-configuration, which can
result in mismatched base pairing (e.g., enol-thymine pairs with guanine
instead of adenine)
 Chemical instability
Depurination: thermal cleavage of the N-glycosidic bond between
deoxyribose and a purine base at body temperature
- An apurinic/apyrimidinic site (AP site) is formed.
- AP sites belong to the most common physiologically occurring DNA
lesions (estimate of > 10,000/day and per cell in eukaryotes).

Dr. E. DILIPAN, AP/Biotech Selvam College of Technology

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Unit-II DNA Replication and Repair

Free radical damage: highly reactive oxygen or hydroxyl radicals that

cause oxidative stress and can chemically alter bases.
- Example: guanosine to 8-oxoguanosine
- Result: During replication, 8-oxoguanosine pairs with adenine and not
cytosine.
Spontaneous deamination of DNA bases causes incorrect base pairing
(mismatching)
Deamination of cytosine to uracil (e.g., as a result of nitrite uptake in
food), which then pairs with adenine
Uracil is usually recognized as an error and replaced with cytosine
through base excision repair.
If this does not occur, uracil pairs with adenine instead of cytosine with
guanine during the next replication.
Deamination of 5-methylcytosine to thymine, which pairs with adenine
instead of guanine.
Deamination of adenine to hypoxanthine
Deamination of guanine to xanthine

Exogenous sources of DNA errors

 Toxic substances
- Alkylating substances
Mechanism of action: methylate or ethylate bases cause incorrect base
pairing, e.g., O6-ethylguanine is formed by the alkylation of guanine and
pairs with thymine instead of cytosine.
Examples: mustard gas, N-nitrosodimethylamine, dimethyl sulfate
- Intercalating substances
Mechanism of action: embedding between the stacked DNA base pairs,
causing replication to stop and increasing the risk of strand breaks.
Examples: ethidium bromide, acridine dye, dactinomycin.
 Radiation
- UV radiation (both UVA and UVB) can result in dimer formation of
neighboring pyrimidine bases (pyrimidine dimers).
- Mainly formation of thymine dimers, linked by a cyclobutane ring.

Dr. E. DILIPAN, AP/Biotech Selvam College of Technology

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Unit-II DNA Replication and Repair

- Dimers create bulky helix distortions that interfere with DNA

replication, which stops replication prematurely, increasing the risk
for further mutations (e.g., BRAF gene mutation in melanoma).
 Ionizing radiation
- Mainly causes dsDNA breaks, but can also cause ssDNA breaks.
- Also results in increased formation of free radicals.

DNA repair mechanism

Type of DNA Phase of Mechanism Associations with

repair cell cycle defective repair
ssDNA repair
Base excision Throughout - Base-specific - Repair of
repair cycle glycosylase (DNA spontaneous or
glycosylase) toxic
recognises and deamination.
removes damaged - Certain
bases (e.g., cancers, e.g.,
deaminated or colorectal
oxidated), creating cancer (MBD4),
an AP site gastric cancer
(apurinic/apyrimidi (NEIL1).
nic).
- AP endonuclease
cuts the DNA
backbone on the 5`
end of the AP site.
- AP lyase cuts the
DNA backbone on
the 3` end of the AP
site.
- DNA polymerase -β
refills the gap.
- Ligase reseals the
strand.
Nucleotide G1 phase - Specific
excision repair endonucleases - Xeroderma
recognize the pigmentosum
damaged area (e.g., (UV radiation
pyrimidine dimers causes
that distort the DNA formation of
helix) of nucleotides pyrimidine
(typically a 12–24 bp dimers)
section).

Dr. E. DILIPAN, AP/Biotech Selvam College of Technology

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Unit-II DNA Replication and Repair

- Oligonucleotide
containing the
damaged region is
excised by
endonuclease.
- DNA polymerase
refills the resulting
gap.
- Ligase reseals the
strand.
DNA mismatch S phase - The newly - Hereditary
repair synthesized strand nonpolyposis
(unmethylated colon cancer.
strand) is
differentiated from
the methylated
parent strand
- Mismatched base
pairs are recognized
by MSH proteins.
- The damaged strand
is cut by
endonuclease.
- Damaged
nucleotides are
removed by
exonuclease.
- DNA polymerase
refills the resulting
gap (no loss of DNA
material).
- Ligase reseals the
strand.
dsDNA break
repair
Nonhomologous Throughout - DNA that has - Ataxia-
end joining the cycle undergone a double- telangiectasia
stranded break is
repaired via DNA
ligase IV.
- Short homologous
sequences called
microhomologies
comprise the single-
stranded tails of the
DNA ends to be
joined.
- Repair is usually
accurate if

Dr. E. DILIPAN, AP/Biotech Selvam College of Technology

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Unit-II DNA Replication and Repair

microhomologies are
compatible
- Nonhomologous end
joining is itself
prone to errors and
mutagenic defects
because no error-
free template is
available.
- Some DNA may be
lost in the process.
Homologous Late S - Repair by - Breast/ovarian
recombination phase or exchanging cancer (due to
repair G2 phase homologous BRCA1 and
segments BRCA2
between two mutations)
DNA molecules - Fanconi anemia
(sister
chromatids)
- The error can be
repaired using
the
complementary
strand from the
intact sister
chromatid.
- Requires a
(nearly) identical
sequence (such
as the
complementary
strand) to serve
as a template for
repair
- No DNA is lost in
the process.

Dr. E. DILIPAN, AP/Biotech Selvam College of Technology

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