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4. Molecular Basis Of Inheritance

-CREATED BY-

PROF. DESHMUKH A. B.

ASST. TEACHER

AGASTI ARTS, COMMERCE AND DADASAHEB RUPWATE SCIENCE JUNIOR COLLEGE, AKOLE

4.4 DNA Replication

Prof. Deshmukh A. B’s. Biology Class

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4.4 DNA Replication :

The DNA molecule regulates and controls all the activities of the cell.
Because of its unique structure,

it is able to control the synthesis of other molecules of the cell.

At the same time when the cell reproduces,

the DNA also should duplicate itself to distribute equally to the daughter cells.

Regulate

&

Control

All Cellular activities

DNA

Cell

Because of its

Unique structure

Able to control the synthesis of other cell molecules

When cell reproduces

Should duplicate itself

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As a carrier of genetic information, DNA has to perform two important functions :
a. Heterocatalytic function :

When DNA directs the synthesis of chemical molecules other than itself, then such functions of DNA are called heterocatalytic functions.

e.g. Synthesis of RNA (Transcription),

synthesis of protein (Translation), etc.

b. Autocatalytic function :

When DNA directs the synthesis of DNA itself, then such function of DNA is called autocatalytic function.
e.g. Replication.

DNA

Heterocatalytic

Autocatalytic

Transcription
Translation

Replication

Being genetic carrier, it performs two important functions

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The process by which DNA duplicates itself is called replication.
Through replication, it forms two copies that are identical to it.
In eukaryotic organisms, replication of DNA takes place only once in the cell cycle.
It occurs in the S- phase of interphase in the cell cycle.
DNA replicates through Semiconservative mode of replication.
The model for Semiconservative replication was proposed by Watson and Crick, on the basis of antiparallel and complementary nature of DNA strands.

Replication

DNA

Daughter DNA

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The process of semicoservative replication is as below:
1. Activation of Nucleotides:

The four types of nucleotides of DNA i.e. dAMP, dGMP, dCMP and dTMP are present in the nucleoplasm.
They are activated by ATP in presence of an enzyme phosphorylase.
This results in the formation of deoxyribonucleotide triphosphates i.e. dATP, dGTP, dCTP and dTTP.
The process is known as Phosphorylation.

dAMP

dGMP

dCMP

dTMP

dATP

dGTP

dCTP

dTTP

+

ATP

Phosphorylase

nucleoplasm

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dAMP = Deoxyadenosine monophosphate (C₁₀H₁₄N₅O₆P )

Adenosine = Adenine + Deoxyribose Sugar

dGMP = Deoxyguanosine monophosphate (C₁₀H₁₄N₅O₇P)

Guanosine = Guanine + Deoxyribose Sugar

dCMP = Deoxycytidine monophosphate (C₉H₁₄N₃O₇P)

Cytidine = Cytosine + Deoxyribose Sugar

dTMP = Deoxythymidine monophosphate (C₁₀H₁₄N₂O₈P)

Thymidine = Thymine + Deoxyribose Sugar

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2. Point of Origin or Initiation point:

It begins at specific point ‘O’ -origin and terminates at point ‘T’.
Origin is flanked by ‘T’ sites.
The unit of DNA in which replication occurs, is called replicon.

‘O’

‘T’

G

T

A

T

A

T

C

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In prokaryotes, there is only one replicon however in eukaryotes, there are several replicons in tandem.
At the point ‘O’, enzyme endonuclease nicks one of the strands of DNA, temporarily.
The nick occurs in the sugar-phosphate back bone or the phosphodiester bond.

Replicon

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3. Unwinding of DNA molecule:

Now enzyme DNA helicase operates by breaking weak hydrogen bonds in the vicinity of ‘O’.
The strands of DNA separate and unwind.
This unwinding is bidirectional and continues as ‘Y’ shaped replication fork.

C G

G C

A T

T A

C G

T A

G C

A T

C G

A T

C G

T A

G C

C G

T A

G C

C G

DNA Helicase

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Each separated strand acts as template.
The two separated strands are prevented from recoiling (rejoining) by SSBP (Single strand binding proteins).
SSB proteins remain attached to both the separated strands so as to facilitate synthesis of new polynucleotide strands.

C G

G C

A T

T A

C G

T A

G C

A T

C G

A T

C G

T A

G C

C G

T A

G C

C G

5’

3’

5’

SSBP

Template Strands

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SSBP

Leading Template

Lagging Template

3’

5’

3’

RNA Primer

Leading Strand

Lagging Strand

(Okazaki fragments)

5’

3’

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Wrong (G≡C)

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4. Replicating fork:

The point formed due to unwinding and separation of two strands appear like a Y-shaped fork, called replicating/ replication fork.
The unwinding of strands imposes strain which is relieved by super-helix relaxing enzyme.

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5. Synthesis of new strands:

Each separated strand acts as mould or template for the synthesis of new complementary strand.
It begins with the help of a small RNA molecule, called RNA primer.
RNA primer get associated with the 3’ end of template strand and attracts complementary nucleotides from surrounding nucleoplasm.

Lagging Template

RNA Primer

Lagging Strand

(Okazaki fragments)

3’

5’

3’

5’

3’

Leading Template

Leading Strand

RNA Primer

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These nucleotide molecules bind to the complementary nucleotides on the template strand by forming hydrogen bonds (i.e. A=T or T= A; G ≡ C or C ≡ G).
The newly bound nucleotides get interconnected by phosphodiester bonds, forming a polynucleotide strand.
The synthesis of new complementary strand is catalyzed by enzyme DNA polymerase.
The new complementary strand is always formed in 5’- 3’ direction.

3’

5’

3’

RNA Primer

5’

3’

A=T

T=A

G≡C

C≡G

A=T

G≡C

A=T

T A

C

A

T

G

T=A

A=T

G≡C

A=T

T=A

G≡C

A=T

C≡G

DNA Polymerase

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6. Leading and Lagging strand:

The template strand with free 3’ end is called leading template and with free 5’ end is called lagging template.
The process of replication always starts at C-3 end of template strand and proceeds towards C-5 end.
As both the strands of the parental DNA are antiparallel, new strands are always formed in 5’ → 3’ direction.
One of the newly synthesized strand develops continuously towards replicating fork is called leading strand.
Another new strand develop discontinuously away from the replicating fork is called lagging strand.

5’

3’

5’

3’

A=T

T=A

G≡C

C≡G

A=T

G≡C

A=T

T A

C

A

T

G

T=A

A=T

G≡C

A=T

T=A

G≡C

A=T

C≡G

3’

Lagging Template

Lagging Strand

(Okazaki fragments)

Leading Template

Leading Strand

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Maturation of Okazaki fragments :

DNA synthesis on lagging template takes place in the form of small fragments, called Okazaki fragments (named after scientist Okazaki).
Okazaki fragments are joined by enzyme DNA ligase.
RNA primers are removed by DNA polymerase and replaced by DNA sequence with the help of DNA polymerase-I in prokaryotes and DNA polymerase-α in eukaryotes.

5’

3’

5’

3’

A=T

T=A

G≡C

C≡G

A=T

G≡C

A=T

T A

C

A

T

G

T=A

A=T

G≡C

A=T

T=A

G≡C

A=T

C≡G

3’

Okazaki Fragments

DNA Polymerase

DNA Ligase

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C≡G

5’

3’

5’

3’

A=T

T=A

G≡C

A=T

G≡C

A=T

T A

C

A

T

G

T=A

A=T

G≡C

A=T

T=A

G≡C

A=T

C≡G

3’

DNA Gyrase

(Topoisomerase)

Finally, DNA gyrase (topoisomerase) enzyme forms double helix to form daughter DNA molecules.

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7. Formation of daughter DNA molecules:

At the end of the replication, two daughter DNA molecules are formed.
In each daughter DNA, one strand is parental and the other one is totally newly synthesized.
Thus, 50% is contributed by mother DNA.
Hence, it is described as semiconservative replication.

C G

G C

A T

T A

C G

T A

G C

A T

C G

A T

C G

T A

G C

C G

T A

G C

C G

G C

A T

T A

C G

T A

G C

A T

C G

A T

C G

T A

G C

C G

T A

G C

C G

Old

New

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Hypotheses about replication (additional info.)

Three hypotheses had been previously proposed for the method of replication of DNA.
In the semiconservative hypothesis, proposed by Watson and Crick, the two strands of a DNA molecule separate during replication.
Each strand then acts as a template for synthesis of a new strand.

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The conservative hypothesis proposed that the entire DNA molecule acted as a template for the synthesis of an entirely new one.
According to this model, histone proteins bind to the DNA, revolving the strand and exposing the nucleotide bases (which normally line the interior) for hydrogen bonding.

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The dispersive hypothesis is exemplified by a model proposed by Max Delbrück, which attempts to solve the problem of unwinding the two strands of the double helix by a mechanism that breaks the DNA backbone every 10 nucleotides or so, untwists the molecule, and attaches the old strand to the end of the newly synthesized one.
This would synthesize the DNA in short pieces alternating from one strand to the other.

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Experimental confirmation :

Semiconservative Replication :

In newly formed DNA molecule, one strand is old (i.e. conserved) and other strand is newly synthesized.
Thus, it is called Semiconservative mode of replication.
It is experimentally proved by Matthew Meselson and Franklin Stahl (1958) by using equilibrium - density - gradient - centrifugation technique.

Old DNA

Daughter DNA

Old Strand

New Strand

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Meselson – Stahl Experiment

1. Meselson and Stahl in 1958 performed an experiment to prove semiconservative nature (mode) of replication.

Meselson and Stahl

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2. They cultured bacteria E.coli in the medium containing ¹⁴N (light nitrogen) and obtained equilibrium density gradient band by using 6M CsCl (Caesium chloride).

(Why Caesium chloride?
Because, it has almost same density as of DNA, i.e., 1.7g/cc)
The position of this band is recorded.

E. Coli grown on culture medium containing ¹⁴N

Centrifugation

¹⁴N labeled DNA

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3. E. coli cells were then transferred to ¹⁵N medium (heavy isotopic nitrogen) and allowed to replicate for several generations.

At equilibrium point density gradient band was obtained, by using 6M CsCl.
The position of this band is recorded.

E. Coli grown on culture medium containing ¹⁵N

Centrifugation

¹⁵N labeled DNA

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4. The heavy DNA (¹⁵N) molecule can be distinguished from normal DNA by centrifugation in a 6M Cesium chloride (CsCl) density gradient.

The density gradient value of 6M CsCl and ¹⁵N DNA is almost same.
Therefore, at the equilibrium point ¹⁵N DNA will form a band.
In this both the strands of DNA are labelled with ¹⁵N.

5. Such E. coli cells were they transferred to another medium containing ¹⁴N i.e. normal (light) nitrogen.

After first generation, the density gradient band for ¹⁴N ¹⁵N was obtained and its position was recorded.
After second generation, two density gradient bands were obtained - one at ¹⁴N ¹⁵N position and other at ¹⁴N position.

6. The position of bands after two generations clearly proved that DNA replication is Semiconservative.

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Grown bacteria in

¹⁵N medium (heavy).

2. Transfer some bacteria to

¹⁴N (Light) medium.

Bacterial growth continues.

3. Take samples after 0 min, 20 min. and 40 minutes.

4. At time0 min, parental DNA was heavy.

5. After 2 generations, half the DNA was intermediate & half was light only.

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Prof. Deshmukh A. B. (Biology)

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Prof. Deshmukh A. B. (Biology)

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THANK YOU

Prof. Deshmukh A. B. (Biology)