DNA replication in prokaryotes occurs through three main steps: initiation, elongation, and termination. Initiation begins at the origin of replication when DnaA and other proteins help separate the DNA strands. Elongation then uses DNA polymerases and other enzymes to bidirectionally copy the DNA in a semiconservative manner, producing both a leading and lagging strand. Termination occurs when the replication forks meet at the terminus region, utilizing proteins like Tus to stop replication.

1. DNA replication in
prokaryotes
By,
Dr. Komal Acharya

2. • DNA replication is a process in which two identical replicas of DNA are produced from one DNA molecule.
• DNA replication in procaryotes is,
 Bidirectional
 Semiconservative

3. • DNA replication in prokaryotes occurs in three steps:
1. Initiation – recognition of the position on a DNA molecule occurs where the
replication begins.
2. Elongation – parent DNA strand gets copied at replication fork.
3. Termination – DNA replication process ends.
 Enzymes/proteins involved in replication process:
Process Enzymes/proteins
Initiation Dna A, SSB (single stranded binding) proteins, Dna B (helicase), Dna
C (helicase loader), Dna G (primase)
Elongation DNA polymerases, SSB, DNA gyrase, DNA ligase
termination Terminus Binding Protein (Tus), DNA topoisomerase IV.

4. 1. initiation:
• DNA replication starts at particular site called origin of replication.
in E.coli, it is called Ori C.
• It spans approx. 245-250 bp of DNA which has two short repeat motifs that
includes, 1. 9 bp sequences called as 9-mer repeats that serve as
binding site for Dna A protein.
2. 13 bp sequences known as 13-mer.
• Replication is initiated when Dna A protein binds to the 9 -mer repeats in
Ori C region and forms an initial complex followed by melting of DNA
duplex at 13-mer repeats in Ori C.
• The separation of double strands of DNA is facilitated by Dna B/ DNA
helicase.
• Ori C also has 11 5’ GATC 3’ repeats in which adenines are methylated that
can initiate the replication.
• Ori C is an AT reach region that makes the breakdown of hydrogen bond
more easier.

5. • This Dna B clamps around each parent strand separated by Dna A. this
binding also require Dna C protein which acts as helicase loader.
• After binding Dna B moves on the DNA strand in the 5’ to 3’ direction and
keep on unwidening the ds DNA.
• Reannealing of the separated DNA strands must be stopped for complete
replication and that is done by SSBs that bind to both the separated strands.
Sequence of events during initiation:
1. Binding of Dna A protein to Ori C region.
2. Loading of DNA helicase.
3. Opening of DNA double helix and binding of primase.
4. Synthesis of RNA primer.
5. Initiation of DNA replication by DNA polymerase.

6. DNA polymerases:
• They catalyze the synthesis of the DNA in 5’ to 3’ direction.
• The DNA polymerases require three things:
1. Template in 3’ to 5’ direction to direct the synthesis of the complementary DNA strand.
2. Primer – small fragments of DNA or RNA
3. dNTPs
Enzyme Function
DNA polymerase I Remove RNA primer through exonuclease activity and replace
it with newly synthesized DNA
DNA polymerase II DNA repair
DNA polymerase III Adds nucleotides to the growing DNA chain in 5’ to 3’ direction
• DNA polymerase III:
It is a holoenzyme which has four components,
1.Two copies of catalytic core enzyme
2.Two copies of dimerization component Tau (Ʈ)
3.Two copies of a ring shaped β-clamp
4.One copy of clamp loader called γ-complex

7. 2. Elongation
• During elongation, DNA polymerases catalyze step by step addition of dinucleotides to the growing strand.

8. • The bacterial replication is a bidirectional replication so, two replication moves in opposite directions and at each replication
fork there is one leading strand and another one is lagging strand.
• Leading strand: strand that is synthesized continuously from a single primer in 5’ to 3’ direction.
• Lagging strand: strand that can be synthesized discontinuously in 3’ to 5’ direction as short DNA fragments from multiple
primers. These short fragments are known as Okazaki fragments and are joined later on by DNA ligase.

9. • DNA polymerase require RNA/DNA primer with a free 3’ OH group annealed to the DNA template.
• DNA primase called Dna G synthesizes short RNA fragments as primers on each template.
• During elongation, this primase is remained associated with the DNA
helicase and this association is called primosome that makes RNA
primers which are then extended by DNA pol III.
• After formation of Okazaki fragments on lagging strand, RNA primers
are removed by DNA pol I. so, gaps are formed in DNA strand that is
filled by DNA pol I. These fragments are ligated together by DNA ligase
that form phosphodiester bond between 3’ OH group of one
fragment and 5’-phosphate group of another fragment.
• A topoisomerase enzyme called DNA gyrase introduce negative
supercoiling into DNA which has nuclease activity and breaks the
phosphodiester bond in a DNA strand.

10. 3. Termination
• Two replication forks meet at a position opposite to the Ori C
region to complete the replication. This position where the
replication terminates is known as terminus region.
• terminus region has 23 bp Ter sequences which are recognized
by sequence specific DNA binding protein called Tus protein
(Terminus Utilization Substance).
• Tus protein has one permissive end and the other non-
permissive end. When the replication fork reaches the
permissive end, Tus-Ter complex dissociates and the fork moves
further. When the fork reaches at non-permissive end,
replication will be terminated.
• In E.coli, Ter sequences are oriented such that, both the forks get
trapped within a short region and the replication terminates.
• after completion of replication, two new identical daughter DNAs
are formed which may be interlocked physically that must be
separated and that is done be topoisomerase IV.