The Replication of DNA in prokaryotes

Chemistry of DNA synthesis

The process of produce new DNA molecules. It is a major role for cell division and growth.
It occurs naturally within cells (in vivo). It can also be replicated artificially in a laboratory.
Nucleotide
The building blocks of DNA. It consist of a sugar (deoxyribose), phosphate group and nitrogenous base
(adenine, guanine, cytosine or uracil, thymine).
ENZYMES
It is a major role in facilitating and regulating DNA synthesis.
It involves main enzymes are:- Helicase, Dna polymerase, DNA primase.
Helicase- It breaks the hydrogen bonds between the two DNA strands, unwinding the double helix.It
creating a replication fork.
DNA polymerase:- Adds new nucleotides to the growing DNA strand, complementary base pairing with
the templates strand.
DNA primase:- It is synthesis short RNA primers to initiate DNA synthesis.
Template strand:- It provides the DNA sequence for building the new DNA strand.DNA polymerase
strictly adheres to base pairing rules (A with T,C with G) to accurate replication.

Artificial DNA synthesis

It creating new DNA molecules in a laboratory.
Oligo synthesis:- The short DNA strand fragments called oligonucleotide are constructed using chemical
building blocks corresponding to each nucleotides.
Fragment assembly:- The synthesized oligos are linked together in a specific order to longer DNA
fragment. In different techniques like restriction enzyme or polymerase chain reaction (PCR)are
employed for assembly.
Verification:-The assembled DNA checked for accuracy using techniques like DNA sequencing to ensure
it matches the desired sequence.

*Enzyme and protein involved in DNA replication

DNA helicase
Topoisomerase
DNA polymerase
DNA ligase
Primase
RNaseH
Telomerase
Sliding clamp and sliding clamp loader
Single strand Binding (SSBs) protein

1)Helicase
Helicase were discovered in E.coli in 1976 and class of enzyme vital in all living organism.
It is known as Helix destabilizing enzyme. They separates the two strands of DNA at the replication fork
behind the topoisomerase. It involved in DNA replication and repair processes.
Function of Helicase
Helicase uses energy from ATP to break the hydrogen bonds holding the base pair together. They can
complementary base pair, allowing the DNA strands to separate.
Which serves as a template for synthesizing new DNA strands.
Mechanism of helicase
Helicase are motor protein that move directionally along the nucleic acid phosphodiester backbone.
They use energy from ATP hydrolysis to separate two hybridized nucleic acid strands.
Importance of helicase
a) DNA replication- They are accurate DNA replication by unwinding the DNA strands.
b) DNA repair- It exposes damaged region for repair enzyme to act.
c) Other processes- involved in transcription, translation, recombination and ribosome biogenesis.
d) Each representing specific processes strand separation is necessary.
e) Approximately1% of eukaryotic gene code for helicase.
f) Human genome encode 95 non reductant helicase including both RNA and DNA helicase.

2)Topoisomerase
a) It was first found by J.O. Wang in the 1970s while working on E.coli , it was the type1
topoisomerase.
b) It help in changing the DNA topology. It can increase or decrease the extent of the unwinding of
DNA.
c) It can also be called DNA strands but there is only on exception topoisomerase.
 Topoisomerase

Type 1

They cut on a single No ATP dependent enzymes

strand of DNA. except Revise gyrase.

Topoisomersa

Type 2

they cuts on both strands of ATP dependent on enzyme.

DNA at once.

d) Topoisomerase are essential enzymes that ensures the proper functioning of DNA by managing its
topological states. Here is a breakdown of there :-

FUNCTION

VARIET
Y

MECHANIS
M IMPORTANCE

 Untangling complex DNA structure:- It can untangle DNA strands that become knotled or
intertwined during cellular processes like replication and transcription.
 Relaxing supercoiled DNA :- DNA naturally adopts a supercoiled structures to save space within
the Nucleus.To allow main role processes to proceed.
 Enabling DNA strand separation:-DNA replication and transcription , topisomerase creates
temporary breaks in the DNA molecule to allow other protein to access and copy the genetic
information.

IMPORTANCE

 Topoisomerase activity is vital for cell survival & proliferation disruptions in topoisomerase
function can lead to DNA damage,
Genetic instability and cell death.

VARIETY

There are two main types of topoisomerase :-

 Type 1topoisomerase : Make single – strand breaks in DNA.
 Type 2topoisomerase: Create double – strand break in DNA.

DNA Polymerase

 DNA polymerase is a group of enzymes crucial for DNA synthesis during synthesis during
replication and repair.

DEFINITION AND IMPORTANCE

 DNA polymerase catalyze the synthesis of DNA molecules from nucleoside triphosphate , the
building blocks of DNA.
 These enzymes are essential for DNA replication , creating two identical DNA duplexes from a
single original DNA strand.

FUNCTION

REPLICATION :-
 DNA POLYMERASE Synthesize DNA during replication.
 It maintains and transfer genetic information across generation information across generation.
 Works in pairs , replication both DNA strands simultaneously.
 Adds deoxyribonucleotides to the 3’ end of the growing DNA strand.
 The DNA strand grows in the 5’ to 3’ direction.
 Adenine pairs with thymine and guanine pairs with cytosine.
 Requires primers to mitrate replication.
 In prokaryotes DNA polymerase III is the primary enzymes in eukaryotes , it is DNA polymerase.

REPAIR
 DNA repair is essential to maintain genome integrity.
 Proof reading.
 DNA replication is not perfect ; errors occurs after every 10^4 to 10^5 Nucleotides added.
 DNA polymerase remove incorrect pairs by exonuclease activity mismatched pairs.
 They proof read by moving one step back and removing mismatched pairs.
 Post – replication repair.
 DNA polymerase participate in repairing damage DNA.

STRUCTURES AND
TYPES
 Most DNA polymerase resemble a hand with active sites.
 Types include :-
 DNA polymerase I :- Remove RNA primer and replace them during lagging strand
synthesis.
 DNA polymerase III :- Main enzymes for replication in proparyotes.
 DNA polymerase (delta):- Main enzymes for replication in eukaryotes.
MECHANISM OF DNA POLYMERASE

 DNA polymerase are pivotal enzymes that help in synthesis of DNA strands , a process
fundamental to the propagation of genetic information.
 There mechanism of action is intricate , ensuring the accurate replication of the genetic code.

PHOSPHORYL GROUP TRANSFER

 The core reaction catalysed by DNA polymerase is the phosphoryl group transfer in this
reaction , the 3’ – OH group of the growing DNA strand acts as a Nucleophile , targeting the α –
phosphorus of the incoming deoxyribonucleoside triphosphate (DNTP) this result in the
formation of a phosphodiester bond and the concurrent release of an inorganic phosphate(pi).

 The enzymatic
MAGNESIUM activity
IONS of DNA
IN THE polymerase
ACTIVE SITE is contingent upon the presence of two magnesium ion
in its active sites these ions facilitate the correct positioning of the reacting groups and stability
the transition of the reaction.

DIRECTIONALITY OF
SYNTHESIS
 DNA polymerase exhibits a strict directionality adding nucleotides exclusively to the 3’ end of the
growing DNA strand , consequently.
 DNA synthesis invariably progresses in the 5’ to 3’ direction notably , DNA polymerase lack the
capability to the initiate DNA strand synthesis de novo; A primer the initial 3’OH group.

BIODIRECTIONAL REPLICATION
It is process by which DNA molecule duplicate during cell division unlike undirection replication,
where replication proceeds in only one direction, biodirectional replication involve synthesizing
DNA in both forward and reverse directions simultaneously.
 Process
1)Origin of replication: DNA replication beings at specific sites origin of replication.
2)Antiparallel strands: DNA strands are antiparallel one strand runs in the 5’-3’
direction.
3)Simultaneous synthesis: During replication, strands of DNA are synthesized
concurring adding nucleotides to the Naxent DNA chain.
4)Leading and lagging strand:- Biodirectional replication result in a leading strand and
lagging strand known as Okazaki fragment.
 Semi- conservative nature –
Each DNA double helix consist of one old parental strand and one new daughter strand.
The semi conservative model ensures genetic continuity.
It is the process by which DNA molecule duplicates itself during cell division. It ensures
that each new DNA double helix consist of one original strand and one newly synthesized
strand.

 SIGNIFICANCE
 Semiconserative replicaton ensure genetic continuity across generation.
 It allows for acurate transmission of genetic information during cell division.

# Discontinuous nature of replication:-

-The lagging DNA strand is synthesized in short discontinuous segment known
as Okazaki fragment.
-While the leading srand is synthesized
Continuously to one directon and lagging
Strand is synthesized in opposite direction in
Fragment.
-The short Okazaki fragment are joined by
DNA ligase.