DNA Replication in Prokaryotes

DNA replication begins at a site termed as the Origin of Replication (OriC). Each bacterial chromosome has only one origin of replication. 

Three types of DNA sequences in OriC are functionally significant.

• AT-rich region
• DnaA boxes
• GATC methylation site

Synthesis of DNA occurs BIDIRECTIONALLY around the bacterial chromosome. The two replication forks eventually meet at the opposite side of the bacterial chromosome. This ends replication process. 

 

Requirements:

Dna A protein: Recognizes OriC sequence, opens duplex at specific sites in origin

Dna B protein (HELICASE): unwinds DNA (requires ATP)

Dna C protein: required for Dna B binding at origin

PRIMASE (Dna G protein): synthesizes RNA primers

SSB protein: binds single stranded DNA

RNA polymerase: facilitates Dna A activity                                      

DNA gyrase (DNA topoisomerase II): relieves torsional strain generated by DNA-binding

Dam Methylase: methylates 5^’ GATC sequence at OriC

DNA ligase: joins broken DNA segments together

Note: In E.coli, there are 4 types of toposiomerases (I to IV); I to III topoisomerases relax DBA by removing negative supercoils. Topoisomerase IV is responsible for separation of catenanes.

DNA polymerase: These are enzymes that catalyze the attachment of nucleotides to synthesize a new DNA strand.

In E.coli, there are five proteins with polymerase activity, namely, Dna Pol I, II, III, IV, and V

Dna pol I and III are involved in normal replication

Dna pol II, IV, and V are responsible for repair and replication of damaged DNA.

Dna Pol I

. composed of single polypeptide

. Removes the RNA primers and replaces them with DNA

. Possess 3’-5’ exonuclease

. Possess 5’-3’ exonuclease – only found in DNA Pol I

  

Dna Pol III

. Responsible for most of the DNA replication

. Composed of 10 different subunits

. Possess 3’-5’ exonuclease

NOTE: DNA polymerases can attach nucleotides only in the 5’ to 3’ direction.

Steps:

DNA replication initiates by the binding of DnaA proteins to the DnaA box sequences. This binding stimulates the cooperative binding of additional ATP-bound DnaA proteins to form a large complex.

DNA helicase separates the two DNA strands by breaking the H-bonds between them. It is an ATP-dependent phase.

This generates positive supercoiling ahead of each replication fork.

DNA gyrase travels ahead of the helicase and alleviates these supercoils.

Single strand binding proteins bind to the separated DNA strands to keep them apart.

The short (10-12 nucleotides) RNA primers are synthesized by DNA primase. 

The short RNA strands start or prime DNA synthesis.

• The leading strand gas a single pair, the lagging strand needs multiple primers.

• They are eventually removed and replaced with DNA.
• The short RNA strands start or prime DNA synthesis.
• The leading strand gas a single pair, the lagging strand needs multiple primers.
• They are eventually removed and replaced with DNA.

The two daughter strands are synthesized in different ways   

1. Leading strand

• One RNA primer is made at the origin.
• DNA Pol III attaches nucleotides in a 5’ to 3’ direction, as it slides towards the opening of the replication fork.

     2. Lagging strand:

• Synthesis is also in the 5’ to 3’ direction.
• Many RNA primers are required, as DNA synthesis takes place in the opposite direction of the opening of the replication fork.
• DNA Pol III uses the RNA primers to synthesize small DNA fragments. These small fragments are called Okazaki fragments in the name of the scientist who discovered it.

Note: RNA Pol I removes the RNA primers and fills the resulting gap with DNA. It uses 5’-3’ exonuclease activity to to digest RNA and 5’-3’ polymerase activity to replace it with DNA. 

• Enzyme complex (DNA Pol I) minus 5’-3’ exonuclease is called Klenow Fragment

Termination of Replication

• On the opposite side of the chromosome to OriC , is a pair of termination sequence called “ter sequence (20 bp).” These are designated as T₁ and T₂
• The protein tus (Termination Utilization Substance) binds to the ter sequence.
• The protein tus, bound to the ter sequence stops the movement of the replication fork.
• DNA replication ends when oppositely advancing forks meet (usually at T₁ or T₂ )
• Finally, DNA ligase covalently links the two daughter strands.
• DNA replication often results in two interwined molecules.
• Interwined circulare molecules are termed catenanes.
• Catenanes are separated by the action of topoisomerase.

Note: Eukaryotic DNA replication is not well understood as bacterial replication. 

Difference between Prokaryotic and Eukaryotic DNA replication

Prokaryotic DNA Replication	Eukaryotic DNA Replication
DNA replication takes place in the cytoplasm	DNA replication takes place in the nucleus
DNA replication takes place in connection with the cell division or during binary fission	It is a complex process that takes place during cell cycle involving different steps. G1, S, G2, M
Single OriC is present	Many OriC are present
The number of nucleotides in OriC is 100-200 nucleotides long or more	Each OriC is 140 nucleotides long
Two replication forks are formed	Many replication forks are formed
Single replicon (It is a unit of DNA in which individual acts of replication take place i.e., it is capable of DNA replication independent of other segments of DNA)	Many replicon present
Initiation of replication is carried out by Dna A and Dna B protein, etc.	Initiation of DNA replication takes place by a multisubunit protein complex
Both leading and lagging strands are synthesized by DNA Pol III	Leading strand is synthesized by DNA Pol delta and lagging strand by DNA Pol alpha
The size of Okazaki fragments is 1000-2000 nucleotides	The size of Okazaki fragment is 100-200 nucleotides long
Replication speed = 200 nucleotides/sec	Replication speed = 100 nucleotides/sec
Termination sequence, called Ter sequence is present	Telomere is the termination site