REPLICATION - EXAM A2Z

REPLICATION

Semi-conservative replication of Chromosomes in eukaryotes:

Autoradiography experiment in Vicia faba, by J.H. Taylor and his co-workers for the study of duplicating chromosomes in the root tip cells were first published in 1957.
They reported that DNA in all the organisms has the inherent capacity of self-replication.
The mechanism of DNA replication is so precise that all the cells derived from a zygote contain exactly similar DNA both in terms of quality and quantity.
The replication takes place in interphase after every cell division.

Theoretically, there may be following three possible modes of DNA replication:

Dispersive Method
Conservative Method
Semiconservative Method

Semiconservative mode is the most accepted of all.

DNA Replication is Semi-Conservative:

Watson and Crick model suggested that DNA replication is semi-conservative.
It implies that half of the DNA is conserved.
Only one new strand is synthesized, the other strand is the original DNA strand (template) that is retained.
Each parental DNA strand serves as a template for one new complementary strand.
The new strand is hydrogen bonded to its parental template strand and forms a double helix.
Each of these strands of the double helix contains one original parental strand and one newly formed strand.

Meselson and Stahl Experiment:

Mathew Meselson and Franklin Stahl proved experimentally that parental strands of a helix are distributed equally between the two daughter molecules.
They made use of the heavy isotope ¹⁵N as a tag to differentially label the parental strands. E. coli was grown in a medium containing ¹⁵N labeled NH₄Cl.

In this way both strands of DNA molecules were labeled with radioactive heavy isotope ¹⁵N in their purines and pyrimidines.
Therefore both strands were heavy or H DNA.
The bacteria were then transferred into a medium containing the common non-radioactive nitrogen ¹⁴N, which is a light medium.
It was found that after one cell division daughter molecules had one ¹⁵N strand the other ¹⁴N strand.
So this is a hybrid molecule, a heavy light of HL.

Fig. Semiconservative replication.

Enzymes Involved In DNA Replication:

Both the prokaryotic and eukaryotic cells contain three types of nuclear enzymes that are essential for DNA replication. These enzymes are nucleases, polymerases and ligases.

(i) Nucleases:

The polynucleotide is held together by phosphodiester bonds.
The nucleases hydrolyse the polynucleotide chain into the nucleotides.
It attacks either at 3′ OH end or 5′ phosphate end of the chain.
The nucleases are of two types.

(a) Exonucleases:

The nuclease that attacks the outer free end of the polynucleotide chain is called exonuclease.
It breaks phosphodiester bonds either in direction 5’→ 3′ or in 3’→ 5′ direction.
The enzyme moves in either cases stepwise along the chain and removes nucleotides one by one.
Thus, the whole chain is digested.

(b) Endonucleases:

The endonucleases attack within the inner portion of one or the double strands.
A nick is made on a double stranded DNA molecule.
However, if the polypeptide chain is single stranded (e.g. in DNA viruses), the attack of endonuclease will render the chain into two pieces.
On double stranded DNA the nick contains two free ends that in turn act as template for DNA replication.
Apart from this, the nicked double helix is distorted due to rotation of free molecules around its intact strand.

(ii) DNA Polymerases:

DNA polymerases carry out the process of polymerization of nucleotides and formation of polynucleotide chains.
This enzyme is called replicase when it replicates the DNA molecules and is inherited by daughter cells.
In prokaryotes, three types of DNA polymerases e.g.
polymerase I (Poly-I),
polymerase II(Pol II), and
DNA polymerase III (Pol III) are found,
whereas in Eukaryotic cells contain five DNA polymerases: α, β, γ, δ, and ε. Polymerase γ is located in mitochondria and is responsible for replication of mitochondrial DNA.
The other four enzymes are located in the nucleus and are therefore candidates for involvement in nuclear DNA replication.
Polymerase β is most similar to E. coli DNA Pol I because its main function is associated with DNA repair, rather than replication. DNA polymerase β is mainly used in base-excision repair and nucleotide-excision repair.
DNA polymerase 𝝳 is the main enzyme for replication in eukaryotes. It also has 3’→5′ exonuclease activity for proofreading. DNA polymerase 𝜶’is main function is to synthesize primers.

(a) Polymerase I (Pol I):

It is discovered by The Kornberg

It is also known as Kornberg polymerase .
It is a single peptide chain with a molecular weight of 109,000 D.
One atom of zinc (Zn) per chain is present, therefore, it is metalloenzyme.
In E. coli, approximately 400 molecules of Pol I are present.
It is roughly spherical of about 65 A° diameters.

Pol I possesses several attachment sites

such as:

(i) A template site for attachment to the DNA template,

(ii) A primer site of about 100 nucleotides contemporary to a segment of RNA on which the growth of newly synthesized DNA occur,

(iii) A primer terminus site containing a terminal 3’OH group at the tip, and

(iv) A triphosphate site for matching the incoming nucleoside triphosphates according to complementary nucleotide of DNA template.

Function:

In E.coli the following three types of functions of Pol I have been found.

Polymerization:

it cannot synthesize a long chain.
It synthesizes only a small segment of DNA in 5′ → 3′ direction
it takes part in repair synthesis.
In E.coli Pol I polymerize the nucleotides at the rate of 1,000 nucleotides per minute at 37°C.

Exonuclease activity:

3’ → 5′ exonuclease activity:

Pol I catalyses the breaking of one or two DNA strands in 3’ → 5′ direction into the nucleotide components
Therefore, it is called 3′ → 5′ exonuclease activity.
Pol I correct the errors made during the polymerization, and edits the mismatching nucleotides at the primer terminus before the start of strand synthesis.
Therefore, the function of Pol I is termed as repair synthesis.

5′ → 3′ exonuclease activity:

Pol I also breaks the polynucleotide chain in 5′ → 3′ direction with the removal of nucleotide residues.
Upon exposure of DNA to the ultraviolet light two adjacent pyrimidines such as thymines are covalently linked forming pyrimidine dimers.
These dimers block the replication of DNA.
Therefore, removal of pyrimidine dimers e.g. thymine dimers (T=T) is necessary.
Through 5′ → 3′ exonuclease activity, Pol I removes pyrimidine dimers.
Secondly, DNA synthesis occurs on RNA primer in the form Okazaki fragments.
Through 5′ → 3′ exonuclease activity Pol I remove RNA primer and seal the gap with deoxyribonucleotides.

(b) Polymerase II (Pol II):

Pol II is a single polypeptide chain (MW 90,000) that shows polymerization in 5’ → 3’ direction of a complementary chain.
It also shows exonuclease activity in 3’ → 5’ direction but not in 5’ → 3’ direction.
The polymerization activity of Pol II is much less than Pol I in E.coli cells.
About 50 nucleotides per minute are synthesized.
E.coli cells contain about 40 Pol II molecules.
The 3′ → 5′ exonuclease activity of Pol II shows that it also plays a role in repair synthesis or DNA damaged by U.V. light just like Pol I.
In the absence of Pol I, it can elongate the Okazaki fragments.
Therefore, Pol II is an alternative to Pol I.

(c) Polymerase III (Pol III):

DNA polymerase III is several times more active than Pol I and Pol II enzymes.
It is the dimer of two polypeptide chains with molecular weight 1,40,000 and 40,000 D respectively.
Pol III is the main polymerization enzyme that can polymerize about 15,000 nucleotides per minutes in E. coll.
Synthesis of a long template also occurs when an auxiliary protein DNA (co-polymerase II) is linked with Pol III and produced Pol III-co Pol II complex.
In addition Pol III also shows 3’→ 5′ exonuclease activity like Pol II.
The 5’→ 3′ exonuclease activity is absent.

(iii) DNA Ligases:

The DNA ligases seal single strand nicks in DNA which has 5’→ 3′ termini.
It catalyses the formation of phosphodiester bonds between 3′-OH and 5′-PO4 group of a nick, and turns into an intact DNA.
There are two types of DNA ligases: E. coli DNA ligase and T4 DNA ligase.
The E. coli DNA ligase requires nicotinamide adenine dinucleotide (NAD+) as cofactor,
whereas T4 DNA ligase uses ATP as cofactor for joining reaction of the nick (Fig 5.19).

DNA Helicase enzyme

This is the enzyme that is involved in unwinding the double-helical structure of DNA allowing DNA replication to commence.
It uses energy that is released during ATP hydrolysis, to break the hydrogen bond between the DNA bases and separate the strands.
This forms two replication forks on each separated strand opening up in opposite directions.
At each replication fork, the parental DNA strand must unwind exposing new sections of single-stranded templates.
The helicase enzyme accurately unwinds the strands while maintaining the topography on the DNA molecule.

SSB proteins:

Bind to the single strands of unwound DNA to prevent reformation of the DNA helix during replication.

DNA primase enzyme

This is a type of RNA polymerase enzyme that is used to synthesize or generate RNA primers, which are short RNA molecules that act as templates for the initiation of DNA replication.

Topoisomerase

This is the enzyme that solves the problem of the topological stress caused during unwinding.
They cut one or both strands of the DNA allowing the strand to move around each other to release tension before it rejoins the ends.
And therefore, the enzyme catalysts the reversible breakage it causes by joining the broken strands.
Topoisomerase is also known as DNA gyrase in E. coli.