DNA Replication: Steps and Mechanism

Deoxyribonucleic acid (DNA) can make an exact copy of itself which is the most important property of DNA. Replication is the process of copying a parental DNA molecule into two daughter DNA molecules. There are three modes of replication of DNA: semi-conservative, conservative, and dispersive.

Semi-conservative model: The two parental strands separate into single strands. Then each strand copies itself, forming one old and one new strand. Each of the strands acts as a template and carries all the information of the original parent DNA molecule.

Conservative model: After the replication, one of the two old strands is conserved.

Dispersive model: The material in the two parental strands is distributed between the two daughter molecules. 

Replication of DNA
Semiconservative model of replication, Source: BROCK Biology of Microorganism

Replication of DNA in prokaryotes

DNA replication in prokaryotes is a semi-conservative type. Double-stranded DNA consists of two strands, each acting as a template for synthesizing the new strand. Then a double-stranded DNA molecule is formed in which one is the new strand, and another is the old strand. It produces two double-stranded daughter molecules arranged in the antiparallel orientation, i.e., 3’- 5’ and 5’-3’ direction. In rapidly growing bacteria like E. coli, the time for replication is 40 minutes.

DNA template is a single strand of DNA having the nucleotide sequences (adenine, guanine, cytosine, thymine) encoded in it. Based on the encoded sequences, nucleotides are added to the next strand of DNA. E.g., if Adenine (A) is encoded in the template DNA, the matching nucleotide Thymine (T) is added to the next strand of DNA (complementary strand).

DNA replication in prokaryotes
DNA replication in prokaryotes

Steps of replication of DNA in prokaryotes

Replication of DNA has three steps: initiation, elongation, and termination.

Initiation

DNA replication occurs from the origin of replication, the single unique nucleotide sequence (or a site). Ori C is the name of the origin site in E. coli. It consists of 245 base pairs (bp). Three repeats of the 13 bp sequence and four repeats of a 9 bp sequence are present. In E.coli, there is the presence of 13-mer region, repeats of a 13 bp (GATCTATTATTTT), and the 9-mer region, four repeats of 9 bp (TTATCCACA). Different enzymes and proteins are responsible for the initiation of replication. They are:

DnaA protein

Four to five DnaA proteins form a single complex and bind to the origin’s four 9 bp repeats. It then recognizes and successively denatures the DNA in the region of the three 13 bp repeats rich in A=T pairs. Then a denatured region is generated, known as the replication bubble.

DnaC protein

In the denatured (unwound) region, DnaC loads the helicase (Dna B protein ) in it.

DNA helicases (or DnaB protein)

Helicase loads on each single-strand of DNA, and the unwinding of the double helix DNA starts in the bi-direction. It then creates the two replication forks. This unwinding process creates the V-shaped structure known as the replication fork. 

Single-stranded binding (SSB) protein

Singe-stranded binding protein only binds to the single-stranded DNA. It prevents the renaturation of the double helix and stabilizes the separated strands. SSB also protects the DNA from the nuclease enzyme (an enzyme that cleaves the ssDNA). 

DNA topoisomerase

As the double-strand starts to separate, supercoiling or super twisting may occur in the region of DNA. Supercoilings are of two types: positive supercoiling and negative supercoiling.

A positive supercoil contains more turns and coil in the direction of the tightening of the coils. Negative supercoil contains fewer turns of the helix and it twists in the direction of the loosening of the coils. DNA topoisomerase resolves the supercoils. 

Type I DNA topoisomerase has both the nuclease (strand cutting) and ligase (strand sealing) activities. It cuts the single strand of the double helix and reseals. It does not require ATP. In E. coli, it relaxes negative supercoils.

Type II DNA isomerase (DNA gyrase) has both nuclease and ligase activities. It cuts both strands in supercoiling. Both the negative and positive supercoils relax. It uses energy from the hydrolysis of ATP.

Elongation

To begin the replication process requires RNA primer. DNA polymerase adds the deoxyribonucleotides to the primer, and the elongation process starts.

Replication of DNA (elongation)
Elongation in DNA replication Source: https://biotechkhan.wordpress.com/2014/10/08/dna-replication-in-prokaryotes/

Primase (DnaG protein)

It makes short RNA primers at the replication origin. These RNA primers are 10-60 nucleotides long with a free 3’ hydroxyl group. DNA polymerase adds deoxynucleotides to the primer. 

DNA polymerase III

It catalyzes the elongation of the DNA chain. Primase synthesizes the RNA primer. DNA polymerase III then binds to the RNA primer, and to extend the primer; it keeps adding the deoxynucleotides. The synthesis of the daughter strand occurs only in the 5’ to 3’ direction because DNA polymerase III can add nucleotides only in the 3’ hydroxyl end of the growing strand. Due to the antiparallel orientation of the DNA, two types of strand are formed.   

Leading strand synthesis: The strand that moves from a 3’ to 5’ direction toward the replication fork is called the leading strand. It progresses forward in the direction of the replication fork and is synthesized continuously. The process begins with the synthesis of the RNA primer in which DNA polymerase adds the deoxynucleotides at the 3’- OH group. 

Lagging strand synthesis: The strand is synthesized in the direction away from the replication fork is called the lagging strand. It is synthesized discontinuously. Synthesis occurs by the formation of the short Okazaki fragments. These Okazaki fragments are in between 100 – 1000 bp. 

DNA polymerase I

RNA primers are removed after forming the Okazaki fragment. Then the DNA polymerase I adds the nucleotides, and the DNA ligase seals the remaining nick.

DNA ligase

DNA ligase catalyzes the formation of the phosphodiester bond. This bond is between a 3’ hydroxyl at the end of one DNA strand and a 5’ phosphate at the end of another strand. 

Proofreading of the newly synthesized DNA

The errors in the replication may lead to the mutation. So proofreading ensures the accuracy of replication. DNA polymerase III checks if the matching of the complementary bases are correct and performs the proofreading activity. 

Termination

Two replication forks in the circular E. coli chromosome meet at a terminus region. It contains multiple copies of a 20 bp sequence called the Ter (for terminus) region. On the chromosome, the arrangement of the Ter sequences are in such a way that it creates the trap for a replication fork. The fork can enter into it but cannot leave it. The Ter sequences are the binding sites for a protein called Tus (terminus utilization substance). The Tus-Ter complex can arrest a replication fork from only one direction. When a replication fork encounters a functional Tus-Ter complex, it stops there, and another fork stops when it meets the first (arrested) fork.

Then, the separation of the two newly made DNA requires the topoisomerase IV. Then during the process of cell division, these separated chromosomes segregate into the daughter cells.   

source: https://www.youtube.com/watch?v=TNKWgcFPHqw

Difference between replication in prokaryotes and eukaryotes

ProkaryotesEukaryotes
Occurs in the cytoplasm of a cellOccurs in the nucleus of a cell
Has one origin of replicationHave several origins of replication
Five polymerases are involved in the replication of prokaryotes: I, II, III, IV, and V.
Function:
I: is involved in the synthesis, proofreading, repair, and removal of RNA primers
II: is also a repair enzyme
III: is a main polymerizing enzyme
IV and V are repair enzymes under unusual conditions.
Five polymerases are involved in the replication of eukaryotes: α, β, γ, δ, ε
Function:
α: a polymerizing enzyme 
β: a repair enzyme
γ: mitochondrial DNA synthesis
δ: main polymerizing enzyme
ε: function unknown    
 
Polymerases are exonucleasesNot all polymerases are exonucleases
Okazaki fragments are 1000-2000 residues long.Okazaki fragments are 150-200 residues long.
No proteins (or histone-like protein) complexed to DNAHistones complexed to DNA
Prokaryotic replication requires DNA gyrase.Prokaryotic replication doesn’t require DNA gyrase.
Two replication forks are formed. Many replication forks are formed in the different replication bubbles. 
DNA is circular.DNA is linear.
Tus-ter complex is present in prokaryotes.Tus-ter complex is absent in eukaryotes.

References:

  • Madigan, M. T., Martinko, J. M., Stahl, D. A., & Clark, D. P. (2011). BROCK Biology of Microorganisms (13th edition). Benjamin Cumming.

Sushmita Baniya

Hello, I am Sushmita Baniya from Nepal. I am a postgraduate student of M.Sc Medical Microbiology. I am interested in Genetics and Molecular Biology.

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