Plasmids are small, circular DNA structures that can replicate independently of the host chromosome. They are commonly found in bacteria and play important roles in processes like drug resistance. Plasmid replication involves the recognition of an origin of replication sequence by plasmid-encoded initiator proteins. This leads to unwinding of the DNA and assembly of a replisome complex. The replication then proceeds bidirectionally via a rolling circle mechanism, where the growing DNA strand displaces the parental strand. Replication terminates once the circular plasmid is completely duplicated.

1. PLASMID
NEERAJA. A
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2. DEFINITION
 a genetic structure in a cell that can replicate
independently of the chromosomes, typically a
small circular DNA strand in the cytoplasm of a
bacterium or protozoan.
 They are autonomously replicating pieces of DNA

3. INTRODUCTION
 Used in the study of replication and DNA repair, due to its
 small size
 Dispensability to host
 Easy manipulation
 Responsible for the evolution of drug resistance & evolution of
complex interaction with animal or plant hosts

4. REPLICATION
 Replication varies depending on
1. The initiation of DNA replication &
2. Leading and lagging strand synthesis are coupled or uncoupled

5. REPLICATION INITIATION
 Replication initiation depends on a sequence – plasmid origin
of replication ( ori ).
 Basic replicon - the minimal sequence that supports
replication, preserving the regulating circuitary.
 Minimal replicon – the minimal portion supporting the plasmid
replication

6. CONTD..
 Rep proteins – plasmid encoded initiators of replication.
 Some theta plasmids rely on host initiation factors for
replication.
 Rep recognition sites consists of direct repeats or iterons.
 Rep binding of ori iterons leads to the formation of
nucleoprotein complex that opens up DNA duplex at A+T rich
segment.

7. CONTD...
 ITERONS- Iterons are directly repeated DNA sequences
which play an important role in regulation of plasmid copy
number in bacterial cells. It is one among the three negative
regulatory elements found in plasmids which control its copy
number

8. DUPLEX MELTING&REPLISOME
ASSEMBLY
 Two types- depending on replicon
1. Dependent on transcription
2. Mediated by trans-acting proteins

10.  Replication in eukaryotes is bidirectional, this type is
unidirectional.
 Ideal example of this type is the circular plasmid of bacteria, as it
happens only in circular genomes.
Initiation
 Initiates by the production of nick on one of the two strands
producing free 3́-OH and 5́ phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins (SSBPs).
Bacterial Plasmid

11. ELONGATION
 For Elongation, the DNA polymerase III
binds to the 3́-OH group of broken strand,
using the unbroken strand as a template.
 The polymerase will start to move in a circle
for elongation, due to which it is named as
Rolling circle model. As the elongation
proceeds, the 5́end will be displaced and will grow
out like a waving thread.

12. DNA Polymerase III
Broken Strand
Origin Point

13.  At the point of termination, the linear DNA molecule is cleaved from
the circle, resulting in a double stranded circular DNA molecule and
a single-stranded linear DNA molecule.
 The linear single stranded molecule is circularized by the action of
ligase and then replication to double stranded circular plasmid
molecule.
Origin Point
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TERMINATION

14. STRAND
DISPLACEMENT

15. MECHANISM
 Parental duplex strand have two single stranded
replication initiation sites ( ssi-A & ssi-B)
 Vertical lines show hybridization between DNA
strands.
 Duplex melting is through RepC and RepA
helicase, allowing two sites to form hair pin.

16. Contd..
 The hair pin base are recognized by RepB', which initiates the
synthesis of RNA primer.
 Extension of 3'-OH of primer by POL-III is assisted by RepA.
 Two D loops are formed as parent strands are displaced and
dissociated from each other,leaving ssDNA intermediates (no H-
bonding).
 Synthesis continues in both direction extending area of D loop
formation.

17. Contd..
 Elongation is completed and termination occurs at ssi sites in which
replication began.
 At this point, the newly synthesized daughter strands are restored.
 Segregation: the two daughter strands are ligated, resulting in two
DNA duplexes each containing a parental strand and a daughter
strand

18. STRAND DISPLACEMENT