MOLECULAR
MECHANISMOF DNA
REPLICATION
Hifsa Zia
MPhil Microbiology
INTRODUCTION
• DNA replication is the process by which a double-stranded DNA molecule is
copied to produce two identical DNA molecules.
• Replication is an essential process because, whenever a cell divides, the two
new daughter cells must contain the same genetic information, or DNA, as
the parent cell.
• The replication process relies on the fact that each strand of DNA can serve as
a template for duplication.
• DNA replication initiates at specific points, called origins. Prokaryotes have
single origin (e.g., OriC in E. coli), while In eukaryotes have multiple origins
per chromosome
• Semiconservative: Each daughter DNA has one parental and one new strand.
Enzymes and proteins involved
in DNA replication
• Helicase Enzyme: Unwinds the double helix by breaking hydrogen
bonds between base pairs.
• Single-Stranded Binding Proteins (SSBs): Prevent reannealing of the
separated strands.
• Topoisomerase (Gyrase in prokaryotes): Relieves supercoiling by
cutting and rejoining DNA strands.
• Primase (RNA Polymerase): Synthesizes a short RNA primer (~10
nucleotides) complementary to the DNA template.
Con…
• DNA Polymerase: Adds nucleotides to the 3’ end of the RNA primer.
• Sliding Clamp (e.g., PCNA in eukaryotes, β-clamp in prokaryotes):
Holds DNA polymerase on the template strand for efficient elongation.
• Ligase: Seals the gaps between Okazaki fragments on the lagging
strand by forming phosphodiester bonds.
• There are three steps of DNA replication
i- initiation
ii- Elongation
iii- Termination
ORIGIN OF REPLICATION
1- INITIATION
• DNA synthesis is initiated at particular points within the DNA strand known as
'origins', which are specific coding regions.
• These origins are targeted by initiator proteins, which recruit more proteins
that help aid the replication process, forming a replication complex around
the DNA origin.
• DNA Helicase unwinds the double helix and exposes each of the two strands,
resulting in results in replication forks growing bi-directionally from the origin.
• Single-Strand Binding Proteins (SSBs) stabilize the unwound DNA and keep
the strands from re-annealing.
• Topoisomerase (e.g., DNA gyrase) prevents supercoiling ahead of the
replication fork.
Con..
• DNA Primase is another enzyme that is important in DNA replication.
It synthesize a small RNA primer, which acts as a 'kick-starter' for DNA
Polymerase at free 3’ OH group.
2- ELONGATION
• DNA Polymerase starts adding nucleotides to the 3’ end of the RNA
primer, synthesizing in the 5’ to 3’ direction.
• Three types of DNA polymerase enzymes are involved
• DNA Polymerase I: Removes RNA primers (using 5’ → 3’ exonuclease
activity) and fills the gaps with DNA.
• DNA Polymerase II: Mainly involved in DNA repair
• DNA Polymerase III: Main enzyme for DNA synthesis. It also Adds
nucleotides to both leading and lagging strands.
Con..
• Leading Strand: Synthesized continuously toward the replication fork.
• Lagging Strand: Synthesized discontinuously away from the fork, in
Okazaki fragments.
• Each Okazaki fragment begins with a new RNA primer.
• Sliding Clamp holds DNA polymerase in place for high processivity.
3- TERMINATION
• The process of expanding the new DNA strands continues until there
is either no more DNA template left to replicate (i.e. at the end of the
chromosome), or two replication forks meet and subsequently
terminate.
• The meeting of two replication forks is not regulated and happens
randomly along the course of the chromosome.
• RNAase H removes the RNA primer that is at the beginning of each
Okazaki fragment.
• DNA Ligase joins fragments together to create one complete strand.
