Introduction to Replication, Replication process in Prokaryotes Proposed models of replication, Semi-conservative method by meselson and sthal

2. DNA REPLICATION IN
PROKARYOTES
Submitted by:
Biyyani Suman
RAM/13-76
Dept. of Agril. Microbiology
Submitted to:
Dr. A. Vijaya Gopal
Associate Professor
Dept. of Agril. Microbiology

3. Introduction to Replication
Replication process in Prokaryotes
Proposed models of replication
Semi-conservative method by meselson and sthal
Topics to be covered………

4. Introduction
 DNA replication, the basis for biological
inheritance, is a fundamental process
occurring in all living organisms to copy their
DNA.
 In the process of "replication" each strand of
the original double-stranded DNA molecule
serves as template for the reproduction of
the complementary strand.
 Two identical DNA molecules have been
produced from a single double-stranded
DNA molecule.

5. DNA has to be copied before a cell divides
DNA is copied during the S or synthesis phase
of interphase
The unit of DNA replication is referred to as a
replicon
Mitosis
-prophase
-metaphase
-anaphase
-telophase
G1 G2
S
phase
interphase
DNA replication takes
place in the S phase.

6.  Replicon is the segment of DNA that is capable of DNA
replication
 Each replicon has an origin and terminus
 Bacterial and viral chromosomes usually contain a single
replicon per chromosome
 In Eukaryotes each chromosome is made up of several
replicons ex: 3500 replicons in 4 chromosomes of
Drosophilla

7.  Replication begins at one site called origin (ori) and
proceeds in both directions around the chromosome. It
is a particular sequence in a genome at which replication is
initiated.
 Origins are A T rich which is important for easy un winding of
the two strands.
 Origin of replication in E.Coli is ori C locus its 245 bp long.
 The site at which replication stops.
 E.coli has two termini one on each strand.
 Termination requires tus gene product , which recognize and
binds to ter sequence and stops replication.
7
Origin of Replication

8. BIDIRECTIONAL REPLICATION
•Synthesis of DNA proceeds bidirectionally
around the bacterial chromosome.
•The chromosome of E.coli phage T7 replicates
in a linear form, and DNA replication begins at
one end.
•Replication produces so called eye structure in
the t7 chromosome
•Two replication forks are formed and progress
in the both the directions away from the origin.
•Replication fork will reach one end of the
chromosome much sooner than the other end &
give rise to Y shaped chromosome.

9. Replication fork
Melting produces two Y shaped forks at origin; one located at
each end of origin.
These forks become replication forks when replication begins
Replisome is the smallest functional unit in the factories and
are responsible for copying one segment of DNA
To begin DNA replication, unwinding enzymes called DNA
helicases cause the two parent DNA strands to unwind and
separate from one another at the origin of replication to form
two "Y"-shaped replication forks.

10. Replication Fork:

11.  The double helix is unwound by the enzymes
helicase , and DNA gyrase
 SSBP (single stranded binding protein) helps keep
strands separated
 DNA polymerase III (pol III) is responsible for most of
DNA synthesis
◦ Adds nucleotides to the 3’ end of the daughter
strand of DNA; DNA synthesis is from 5' to 3'
◦ Requires RNA primers as a guide for synthesis
 RNA primers are made by the enzyme primase
 DNA polymerase I: involved in proof reading and
DNA repair
 DNA ligase: involved in connected ends of replicated
DNA together
11

12. ENZYMES FOR REPLICATION

13. Replication process in Prokaryotes
DNA replication includes:
◦ Initiation – replication begins at an origin
of replication
◦ Elongation – new strands of DNA are
synthesized by DNA polymerase
◦ Termination – replication is terminated
differently in prokaryotes and eukaryotes

14. The main events involved in replication
initiation are
Recognition of origin
DNA melting
Stabilization of single strands
Assembly of primosome at the two forks
produced
Start synthesis of two daughter strands

15. I. DnaA recognizes oriC seq.
and opens duplex at specific
sites.
Denaturation of DNA at oriC
requires a histone-like HU
protein and ATP
II. DnaB helicase (hexamers)
binds to unwound DNA
region. Binding of DnaB to
individual strands also
requires DnaC.
III. DNA gyrase unwinds DNA
IV. SSBs bind to single stranded
DNA
v. DnaG primase synthesizes
short RNA primers.
Proteins for initiation

17. Initiation of Replication at oriC
DNA replication is initiated by the binding of
DnaA proteins to the DnaA box sequences
– causes the region to wrap
around the DnaA proteins and
separates the AT-rich region

18. Uses energy from ATP to
unwind the duplex DNA
SSB
SSB SSB
SSB

19. 19

20. INITIATION IN DETAIL
 2-4molecules of DnaA bind oriC results in folding of
DNA around the aggregate & melting starts
 An aggregate having 6 molecules each of DnaB and
DnaC binds to each of the three separate single-standard
regions produced by DnaA
 Agregate eventually displaces DnaA and DnaC loads
DnaB
 DnaB funtions as Helicase and begin unwinding and
Gyrase provides a swivel
 SSBP bind to single strand regions and stabilise them
 One DnaB hexamer binds to each of the two folks
produced by unwinding at the origin

21.  Once replication fork is generated primosome
assembles the origin, and initiates primer
synthesis(priming)
 Priming occurs only once and at origin for the
replication of leading strand
 Priming occurs repeatedly at intervals of 1000 to
2000 bases in case of lagging strand
 The primosome moves along the single standard
region
 When the primosome reaches a site at which a
priming can occur ,it synthesises an RNA primer
and the primer sponsors synthesis of a new
okazoki fragments

22. Melting of DNA by DnaA
Release of DnaB at the forks by DnaC
Helicase action of DnaB
Swivel action of DNA Gyrase
Activation of primase DnaG by DnaB
Activation of DNA polymerase to start replication

23. B) Elongation
Requires at least 7 proteins
a) SSBs bind to ssDNA
b) DnaB helicase unwind DNA,
primosome
c) Primase RNA primer synthesis
d) DNA pol. III new strand elongation
e) DNA pol. I remove primers & fills
gaps
f) DNA ligase seals nicks
g) DNA gyrase supercoiling

24. Parental DNA
DNA pol III
Leading strand
Connecting
protein
Helicase
Lagging strandDNA
pol III
Lagging
strand
template
5
5
5
5
5
5
3 3
3
3
3
3

25. Subunit Function
α DNA polymerase
ε 3’ – 5’ Exonuclease
θ stimulates 3’ – 5’ exonuclease
τ dimerises core and activates helicase
γ binds ATP
δ & ψ Unknown
χ Removal of primase
β Sliding clamp
Catalytic
core
Clamp loader

26. A “clamp loader:”
complex is required
to get the clamp onto
the DNA

27. Elongation in detail
Replisome complex:
• The entire DNA-synthesizing complex at each
replication fork, which also includes
topoisomerase , helicase, and primase, is referred
to as a replisome
• Replication activity is due to the DNA
polymerase III of Replisome.
• E.coli has 10 molecules of DNA polymerase
enzymes

28.  A single holo enzyme molecule functions at one replication
fork.
 Each holo enzyme molecule has 2 catalytic cores, one
catalyses the replication of leading strand and other
catalyses lagging strand.
 In case of leading strand the catalytic core extends the
primer one nucleotide at a time.
 DnaB progressively unwinds the duplex and the replication
fork moves along
 Replication of lagging strand will be after some time.
 When DnaB associated with the advancing fork reaches a
site suitable for priming,it activates DnaG to synthesize a
primer in the normal 5’-3’ direction i.e, towards oriC

29. Theodor Hanekamp © 2003 8
DNA polymerase III holoenzyme


 
’

 
 
Core (
Linker
protein
Clamp
loader
“asymmetric dimer”
Source: “Model after A.Kornberg and T.Baker”,
adapted from Stryer
Polymerase
activity
3’- 5’
exonuclease
processivity
Core (

30.  When the primer becomes10-14bases the other core begins
to elongate in 5’-3’ direction
 The lagging strand is in effect , pulled up by the replisome
in the process of replication
 When the replisome reaches 5’-end of primer of previous
okazaki fagment it stops replication and dissociates from
the lagging strand
 Mean while DnaB continues to move forward with the
replication fork
 When it reaches the appropriate site, it again induces
primer synthesis by DnaG and the eents described above
takes place again

31. Simultaneous synthesis of leading and lagging strands:
• DNA is synthesized only in the 5’-3’ direction and never in the 3’-5’
direction for the complementary strand
• In the 3’-5’ template strand the DNA synthesis made in the 5’-3’
direction in a continuous way is called the leading strand
• On the other template short pieces of DNA ( ~1000 nucleotides
long) in the 5’- 3’ direction are made and the pieces are joined
together.
• The small fragments of DNA are called Okazaki fragments, after
their discoverer, R. Okazaki.
• The new DNA is made by this discontinuous method is called the
lagging strand
• The leading and lagging strands are synthesized simultaneously by a
single dimeric DNA polymerase III complex

32. 32

33. Simultaneous synthesis of
both DNA strands
Source: Stryer

34. DNA Chain extension:
• The primer is then extended by DNA Pol III
• DNA Pol synthesizes DNA for both the leading and
lagging strands
• After DNA synthesis by DNA Pol III DNA pol I
uses its 5’-3’ exonuclease activity to remove the
primer and then fills the gaps with a new 5’ – 3’
exonuclease activity
• Finally, DNA pieces are joined together by the
DNA ligase

35. DNA ligase seals the gaps between Okazaki fragments with a
phosphodiester bond

36. • The sequence that stop the movement of
replication fork are identified as ‘ter’elements
• These are 23 bp consensus sequences that provide
the binding site for the ‘tus’gene, a 36 kD Protein
needed for the termination.
• Tus protein binds to ter elements and stops Dna B
from unwinding DNA.
• Leading strand replicated upto ter elements and
lagging strand replication stopped 50-100bp
before the ter elements.
C) Termination:

37. Origin of replication
Overview
Leading
strand
Leading
strand
Lagging
strand
Lagging strand
Overall directions
of replication
Template
strand
RNA primer
for fragment 1
Okazaki
fragment 1
RNA primer
for fragment 2
Okazaki
fragment 2
Overall direction of replication
3
3
3
3
3
3
3
3
3
3
3
3
5
5
5
5
5
5
5
55
5
5
5
2
2
2
1
1
1
1
1
2
1

38.  In the late 1950s, three different mechanisms were proposed
for the replication of DNA
Conservative model
 Both parental strands stay together after DNA
replication
Semi-conservative model
 The double-stranded DNA contains one parental and one
daughter strand following replication
Dispersive model
 Parental and daughter DNA are interspersed in both
strands following replication
Proposed Models of DNA Replication

39. Semi-conservative model
The double-stranded
DNA contains one
parental and one
daughter strand
following replication
Conservative model
Both parental
strands stay
together after
DNA replication
Dispersive model
Parental and daughter
DNA are interspersed in
both strands following
replication

41. Expt by meselson and sthal:
•Bacterial (E coli) DNA is placed in a media containing heavy
nitrogen(N15), which binds to the DNA, making it identifiable.
•This DNA is then placed in a media with the presence of N14 and
left to replicate only once. The new bases will contain nitrogen 14
while the originals will contain N15.
•The DNA is placed in test tubes containing caesium chloride
(heavy compound) and centrifuged at 40,000 revolutions per
minute.
•The caesium chloride molecules sink to the bottom of the test tubes
creating a density gradient. The DNA molecules will position at
their corresponding level of density (taking into account that N15 is
more dense than N14)
•These test tubes are observed under ultraviolet rays. DNA appears
as a fine layer in the test tubes at different heights according to their
density.

42. 1958: Matthew Meselson & Frank Stahl’s Experiment
Semiconservative model of DNA replication

43. Bacteria
cultured in
medium with
15N (heavy
isotope)
Bacteria
transferred to
medium with
14N (lighter
isotope)
DNA sample
centrifuged
after first
replication
DNA sample
centrifuged
after second
replication
Less
dense
More
dense
Predictions: First replication Second replication
Conservative
model
Semiconservative
model
Dispersive
model
21
3 4
EXPERIMENT
RESULTS
CONCLUSION