DNA Replication - Exam A2Z

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.

After the second cell division, out of four molecules, two DNA molecules contained (LL).
The other two were hybrid molecules (HL).
This proves that during replication, one parent strand is conserved and the other new strand is synthesized.
Thus DNA replication is a semi-conservative process.

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 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 eukaryotes three or four polymerases termed as α, β and ү polymerases and mitochondria (mt) DNA polymerase are present.

(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 pol III is much more complex than DNA pol I.
It has ten types of subunits viz a. Β, Υ, ε, θ, τ, δ, δ’, X, ψ,
The polymerization and proofreading activities are functions of a and ε (epsilon) subunits, respectively.
The θ subunit associates with a and ε to form a core polymerase, which can polymerize DNA but with limited processivity.
Two core polymerases can be linked by another set of subunits, a clamp-loading complex or y complex, consisting of five subunits, τ₂ y δ, δ’.
The core polymerases are linked through the t (tau) subunits.
Two additional subunits x (chi) and ψ (psi), are bound to the clamp-loading complex.
The entire assembly of 13 proteins subunits is called DNA polymerase III.
DNA polymerase III can polymerize DNA but with low processivity.
The increase in processivity is provided by the addition of the four ß-subunits.
This association of DNA pol III with the B-subunits converts this enzyme into DNA pol III holoenzyme.
The B-subunits associate in pairs to form donut shaped structures that encircle the DNA and act like clamps.
Each dimer associates with a core subassembly of polymerase III and slides along the DNA as replication proceeds.
The ß-sliding clamp prevents the dissociation of DNA pol III from DNA, thus increasing processivity.
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.
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 .

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.

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.

Telomerase

This is an enzyme found in eukaryotic cells that adds a specific sequence of DNA to the telomeres of chromosomes after they divide, stabilizing the chromosomes over time.

DNA-Replication– Process

Stages of Replication

The DNA synthesis or replication can be divided into three stages

(1) Initiation

(1) Elongation

(3) Termination

(i) Initiation :

In E coli replication is origin a specific site which is called ori C.
It is highly conserved sequence which is 245 bp long. .
In this sequences there are three repeats of 13 bp AT rich sequence and four repeats of a 9 bp sequence.

At least, nine different enzymes/ proteins participate in the initiation phase
A single complex of four to five Dna A protein molecules binds to the four 9 bp repeats in the ori C
DnaA protein forms a complex of 30-40 molecules, each bound to an ATP molecule and bacterial histone like proteins around which the OriC DNA becomes wrapped
This process cause negatively supercoiled in DNA.
This melting (unwoundıng) of three 13 bp AT-rich repeat sequences.
After this Dna B with the help of Dna C protein load on DNA.
It is a hexamer ring structure two ring.
The two ring of Dna B, and DNA C complex loaded on to each DNA strand and unwinds the DNA bidirectionally by using energy of ATP hydrolysis to move into and melt double stranded DNA
The single stranded bubble created in this way is coated with single strand binding protein (SSB) to protect it from breakage and prevent DNA renaturation.
The enzyme DNA primase then attaches to the DNA and Synthesizes a short RNA primer to initiate synthesis of the leading stand of the first replication fork
Bidirectional replication then follows

Unwinding :

In replication DNA helicases travel along the template strands to open the double helix for copying the nucleotide on DNA template
In addition to Dna B. a second DNA helicase may bind to the other strand to assist unwinding
In a closed circular DNA molecule, removal of helical tums at the replication forks leads to the introduction of additional tums in the rest of the molecule in the form of positive supercoiling
Although the natural negative supercoiling of circular DNA partially compensates for this, it is insufficient to allow continued progression of the replication forks
This positive supercoiling must be relaxed continuously by the introduction of further negative supercoils by type Il topoisomerases (eg DNA gyrase)

(ii) Elongation :

The elongation phase of replication includes two distinct but related events,
leading strand synthesis and lagging strand synthesis
Leading strand synthesis is more simple and begins with the synthesis of short (10 – 60 nucleotides) RNA primer by enzyme primase (Dna G protein)
at the DNA- Replication Deoxyribonucleotides are added to this primer by DNA pol III.
Leading strand synthesis now proceeds continuously keeping pace with the unwinding of DNA at the replication fork
Lagging strand synthesis (DNA polymerase Ill)

Synthesis of Okazaki fragments.

Lagging strand synthesis in short Okazaki fragments
First, a RNA primer is synthesized by primase, and as in leading strand synthesis,
DNA pol III binds to the RNA primer and adds deoxyribonucleotides.
This event seems simple but in fact it is complex, owing to coordination of leading and lagging strand synthesis. both strands are produced by a single asymmetric DNA pol III dimer,
one half synthesising lagging and other half leading strand synthesis.
The DNA of lagging strand is looped so that the two points of polymerization come together ensuring simultaneous synthesis of two strands and at the same rate.
The synthesis of okazaki fragments on the lagging strand involves many enzymes and proteins.
The Dna B helicase and Dna G primase constitute a functional unit within the replication complex, called the primosome.
DNA pol III uses one set of its core subunit (core polymerase) to synthesize the leading strand continuously while the other set of core subunits cycles from one Okazaki fragment to the next on the looped lagging strand.
The Dna B helicase unwinds the DNA at the replication fork as it travels along the lagging strand template in 5′ -3′ direction.
DNA primase occasionally associates with Dna B helicase and synthesizes a short RNA primer A new B sliding clamp is then positioned at the primer by the clamp loading complex of DNA pol III
When synthesis of Okazkı fragment has been completed, replication halts and the core subunits of DNA pol III dissociate from their B sliding clamp (and from the completed Okazaki fragment) and associate with the new clamp.
This initiates synthesis of a new Okazaki fragment.
The entire complex responsible for the co ordinated DNA synthesis of two strands at the replication fork is a replisome
The replisome promotes rapid DNA synthesis, adding – 1000 nucleotides to each strand.
Once an Okazaki fragment has been completed, its RNA primer is removed and replaced with DNA by DNA pol I, the nick is sealed by DNA ligase.

(iii) Termination :

Finally, the two replication forks of the circular E coli chromosome meet at a terminus region containing multiple copies of a 20 bp sequence called Ter (terminus).
The Ter sequences are arranged on the chromosome to create a sort of trap that a replication fork can enter but cannot leave.
The Ter sequence act as binding sites for tus protein (terminus utilization substance)
The tus-ter complex can arrest a replication fork from only one direction Only one Tus-Ter complex per replication cycle performs
Replicated DNA circles linked in this way are called catenanes and their separation requires topoisomerases IV.
The separated chromosomes then seggregate into daughter cells at cell division.

DNA Replication is Semi-Conservative: