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Course : PGPathshala-Biophysics

Paper 10 : Techniques used in Molecular Biophysics II

Module 09 : DNA Replication Models, Mechanisms

Content Writer: Dr. Jayanth Kumar, AIIMS, NEW DELHI

DNA is the genetic material of eukaryotes and some prokaryotes including bacteria. Bacterial

DNA is double stranded and usually free of other proteins. On the contrary, eukaryotic DNA

is wound into complicated secondary and tertiary structures. It is also wound around histone

proteins which restrict or allow access to the DNA for transcription adding another layer of

complexity to gene expression regulation.

DNA structure:

The presence of nucleic acids in the nuclei of pus cells was demonstrated by Friedrich

Miescher in 1869. He named the acidic substance as 'nuclein' which referred to a mixture of

nucleic acids and protein. DNA was established as the genetic material by the Hershey-Chase

and the Mcleod-Avery-Macarty experiments. Subsequently, the first x-ray diffraction

experiments of DNA were carried out by Rosalind Franklin and Maurice Wilkins (1950)

following which the double helical structure of DNA was elucidated by James Watson and

Francis Crick (1953).

The DNA backbone is made of nucleotides.

Nucleotide includes a deoxysugar (deoxyribose)

which is connected to a nitrogenous base which can

either be a purine (guanine or adenine) or a

pyrimidine (cytosine or thymine). Each nucleotide is

linked to the other by a 5'-3' phosphodiester bond. In

the double strand, Adenine usually base pairs with

its complementary nucleotide Thymine by hydrogen

bonding and Cytosine pairs with Guanine. The

hydrophilic sugar moieties are towards the outside of

the double stranded DNA (dsDNA) chain while the

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hydrophobic bases are stacked in the centre. This is called base stacking interactions. DNA

has an absorption maximum at 260 nm. Due to the base stacking interactions, the absorbance

of dsDNA is much lower than that of the corresponding single stranded DNA (ssDNA). This

is called the hypochromic effect.

DNA Replication:

DNA replication is the biological process of producing two identical copies of DNA from one

original molecule. Replication is the basis for biological inheritance. Since DNA is made up

of a double helix of two complementary strands during replication, these strands are

separated. Each strand of the original DNA molecule then serves as a template for the

production of a daughter strand.

Models of DNA Replication:

The Watson and Crick DNA model implies an elegant mechanism for replication:

a. Unwind the DNA molecule.

b. Separate the two strands.

c. Make a complementary copy for each strand.

Three possible models were proposed for DNA replication:

a) Conservative model proposed both strands of one copy would be entirely old DNA,

while the other copy would have both strands of new DNA.

b) Dispersive model was that dsDNA might fragment, replicate dsDNA, and then

reassemble, creating a mosaic of old and new dsDNA regions in each new chromosome.

c) Semiconservative model is that DNA strands separate, and a complementary strand is

synthesized

for each, so that

sibling chromatids

have one old and one

new strand.

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The correct model of DNA replication was established by the Messelson and Stahl

experiment. The experiment made use of heavy Nitrogen (15N) and light Nitrogen (14N) to

label DNA. E.Coli were grown in 15N containing media for several generations and then

shifted to 14N containing media. When DNA is extracted from these cells and centrifuged on a

salt density gradient, the DNA separates out at the point at which its density equals that of the

salt solution. In the first generation, DNA contained a mixture of 15N and 14N which proved

that DNA replication is semiconservative.

DNA replication in prokaryotes

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In general, DNA is replicated by: a) uncoiling of the helix b) strand separation by breaking of

the hydrogen bonds between the complementary strands and c) synthesis of two new strands

by complementary base pairing. Replication begins at a specific site in the DNA called the

origin of replication (ori).

DNA replication is bidirectional from the origin of replication. DNA replication occurs in

both directions from the origin of replication. To begin DNA replication, unwinding enzymes

called DNA helicases cause the two parent DNA strands to unwind and separate from one

another at the origin of replication to form two "Y"-shaped replication forks. Helix

destabilizing proteins (Single strand binding proteins: SSBs) bind to the single-stranded

regions so the two strands do not rejoin.

Coiling of the dsDNA during the unwinding of the strands at the replication fork leads to

formation of supercoils. Enzymes called topoisomerases produce breaks in the DNA and then

rejoin them in order to relieve the stress produced by supercoiling. There are two type of

topoisomerases: Type I which create a nick in a single strand of DNA and relieve stress, Type

II when a double strand break is created, stress is released followed by resealing of the nick.

As the strands continue to unwind in both directions around the entire DNA molecule, new

complementary strands are produced by the hydrogen bonding of free DNA nucleotides with

those on each parent strand. As the new nucleotides line up opposite each parent strand by

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hydrogen bonding, enzymes called DNA polymerases join the nucleotides by way of

phosphodiester bonds. The nucleotides lining up by complementary base pairing are

deoxynucleoside triphosphates (dNTPs). As the phosphodiester bond forms between the 5'

phosphate group of the new nucleotide and the 3' OH of the last nucleotide in the DNA strand,

two of the phosphates are removed providing energy for bonding.

DNA polymerase

DNA polymerases have the property that they can only add nucleotides at the free 3' end.

Hence DNA polymerases can extend the daughter strands in the 5' to 3' direction. The

template strand is therefore read in the 3' to 5' direction. So the two strands of DNA cannot be

replicated the same way. The two strands are antiparallel: one parent strand - the one running

3' to 5' is called the leading strand can be copied directly down its entire length and the other

parent strand - the one running 5' to 3' is called the lagging strand must be copied

discontinuously in short fragments – Okazaki fragments of around 100-1000 nucleotides each

as the DNA unwinds.

DNA polymerase enzymes cannot begin a new DNA chain from scratch. It can only attach

new nucleotides onto 3' OH group of a nucleotide in a preexisting strand. To start the

synthesis of the leading strand and each DNA fragment of the lagging strand, an RNA

polymerase complex called a primosome or primase is required. The primase is capable of

joining RNA nucleotides without requiring a preexisting strand of nucleic acid - this forms an

RNA primer with a free 3' OH group where DNA polymerase can add nucleotides further.

After a few nucleotides are added, primase is replaced by DNA polymerase. DNA polymerase

can now add nucleotides to the 3' end of the short RNA primer. The primer is later degraded

and filled in with DNA.

There are three DNA polymerases found in bacteria: I, II and III. All of them have 5' to 3'

DNA polymerase activity. DNA polymerase I also has 5'-3' exonuclease activity as well as 3'-

5' exonuclease activity. DNA polymerase I is involved in removal of RNA primers after

replication as well as in proofreading of DNA.

DNA polymerase II is involved in repair of damaged DNA. It has 3' → 5' exonuclease

activity. However there are multiple proofs that this is not the main polymerase involved in

DNA Replication.

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A. E.Coli Strains lacking the gene show no defect in growth or replication.

B. Synthesis of Pol II is induced during the stationary phase of cell growth - a phase in which

little growth and DNA synthesis occurs.

C. Pol II has a low error rate but it is much too slow to be of any use in normal DNA

synthesis.

DNA Polymerase III is the main replicative polymerase in bacteria. It has 3' → 5' exonuclease

proofreading ability. This enzyme is highly processive and catalyses polymerization at a high

rate.

There are two forms of the enzyme. The core enzyme - consists of only those subunits that are

required for the basic underlying enzymatic activity: alpha, epsilon and theta. The

holoenzyme is the fully functional form of an enzyme, complete with all of its necessary

accessory subunits. The DNA polymerase III holoenzyme consists of the core enzyme, the β

sliding clamp and the clamp-loading complex. A DNA clamp, also known as a sliding clamp

serves as a processivity-promoting factor in DNA replication. The clamp-polymerase protein–

protein interactions are stronger and more specific than the direct interactions between the

polymerase and the template DNA strand. The presence of the sliding clamp dramatically

increases the number of nucleotides that the polymerase can add to the growing strand per

association event. The presence of the DNA clamp can increase the rate of DNA synthesis up

to 1,000-fold compared with a non-processive polymerase.

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Initiation of replication

Replication starts when DNA at the origin of replication denatures to expose the bases,

creating a replication fork. Replication is usually bidirectional from the origin. E. coli has one

origin, oriC, which has:

A minimal sequence of about 245 bp required for initiation.

Three copies of a 13-bp AT-rich sequence.

Four copies of a 9-bp sequence.

The following sequence of events occur in E. coli initiating DNA synthesis: Initiator proteins

attach. E. coli’s initiator protein is DnaA (from the dnaA gene). DNA helicase (from dnaB)

binds initiator proteins on the DNA, and denatures the AT-rich region using ATP as an energy

source. DNA primase (from dnaG) binds helicase to form a primosome, which synthesizes a

short (5–10nt) RNA primer. Following the formation of the RNA primer, DNA polymerase III

takes over to polymerize the leading and the lagging strands. At the end of replication, the

RNA primer is replaced by DNA by the activity of DNA polymerase I.

Termination of Replication takes place when the replication fork encounters certain

termination sequences which trap the replication fork from moving back. The two forks

moving from the opposite ends meet and replication is complete. The concatenated DNA

strands are separated by DNA topoisomerase IV, which is a Type II topoisomerase.

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Fidelity of DNA replication

DNA replication exhibits a high degree of fidelity. Mistakes during the process are extremely

rare. DNA pol III makes only one mistake per 108 bases made. There are several reasons why

fidelity is high:

Instability of mismatched pairs

Configuration of the DNA polymerase active site

Proofreading function of DNA polymerase

Instability of mismatched pairs: Complementary base pairs have much higher stability than

mismatched pairs. This feature only accounts for part of the fidelity: It has an error rate of 1

per 1,000 nucleotides

Configuration of the DNA polymerase active site: DNA polymerase is unlikely to catalyze

bond formation between mismatched pairs. This induced-fit phenomenon decreases the error

rate to a range of 1 in 100,000 to 1 million

Proofreading function of DNA polymerase: DNA polymerases can identify a mismatched

nucleotide and remove it from the daughter strand. The enzyme uses its 3’ to 5’ exonuclease

activity to remove the incorrect nucleotide. It then changes direction and resumes DNA

synthesis in the 5’ to 3’ direction

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DNA Replication in Eukaryotes:

The basic principle behind DNA replication in eukaryotes is similar to that of prokaryotes.

However there are some key differences. There are multiple origins of replication unlike

prokaryotes which have only one origin of replication. For example, the human genome has

around 30000 origins of replication. Eukaryotes have around 15 DNA polymerases. DNA

polymerase α functions as the primase creating the RNA primer required to initiate

replication. DNA polymerase β is involved in proofreading and DNA repair. DNA

polymerase δ is the main polymerase involved in replication of the leading and the lagging

strands. Eukaryotes have mitochondria which carries its own genetic material. DNA

polymerase γ carries out replication and repair of mitochondrial DNA. Other polymerases are

not well characterized. Not all the DNA polymerases are exonucleases in eukaryotes.

DNA replication in Eukaryotes is synchronized with the progression of the cell cycle. During

the S phase of the cell cycle, DNA synthesis begins. The function of helicase is taken over by

the MCM (Minichromosome maintenance) group of proteins. Proteins like Cdc6 and Cdt

prevent replication from progressing until the cyclin dependent kinases become active and

phosphorylate them thus linking cell cycle changes to DNA replication.

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In prokaryotes replication proceeds at about 1000 nucleotides per second, and thus is done in

no more than 40 minutes. In Eukaryotes replication takes proceeds at 50 nucleotides per

second, and is completed in 60 minutes.

Telomerase

The RNA primer of the last Okazaki fragment cannot be replaced by DNA because of the

absence of a free 3'-OH group to add nucleotides. This results in an overhang at the ends of

chromosomes. Overhangs are good substrates for exonucleases. To prevent this the cell

utilizes the enzyme telomerase, also called terminal transferase. This is a ribonucleoprotein

that adds a species-dependent telomere repeat sequence to the 3' end of telomeres. A telomere

is a region of repetitive sequences at each end of eukaryotic chromosomes. Telomeres protect

the end of the chromosome from DNA damage or from fusion with neighbouring

chromosomes.

Telomerase is a reverse transcriptase enzyme that carries its own RNA molecule (e.g., with

the sequence "CCCAAUCCC" in vertebrates) which is used as a template when it elongates

telomeres. As more and more repeats are added, DNA polymerase III starts adding more

Okazaki fragments on the complementary strand. Thus the ends of the chromosome are

prevented from damage and loss of genetic material.

Telomerase is highly active in normal stem cells and most cancer cells. It is normally absent

from, or at very low levels in, most somatic cells. This explains the finite lifespan of somatic

cells since they keep losing genetic material from the ends of chromosomes and are not able

to replace them.

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Summary:

DNA

replication is

the biological

process of

producing two

identical copies

of DNA from one

original

molecule.

DNA

replication is

semiconservative

It is carried out by multiple proteins including DNA polymerase III in prokaryotes

Only one origin of replication is present in prokaryotes and multiple are present in

eukaryotes

DNA replication is possible only in the 5' to 3' direction: Hence two different types of

replication take place in the two strands: Leading strand and lagging strand synthesis

The short fragments formed in the lagging strand: Okazaki fragments

Telomerase is a reverse transcriptase which helps to protect the ends of chromosomes.

DNA replication is highly synchronized to the cell cycle in eukaryotes

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The β clamp helps in increasing the processivity of DNA polymerase

Termination sequences and DNA topoisomerase help in the termination of replication.