The document summarizes key aspects of DNA replication in bacteria. It describes how the leading strand is synthesized continuously in the 5' to 3' direction, while the lagging strand is synthesized discontinuously in short Okazaki fragments in the 3' to 5' direction. It also discusses the roles of the helicase, which unwinds the DNA, and the single-stranded binding protein, which coats and protects the exposed single strands. Primers are also required to provide 3' OH ends for DNA polymerase to begin synthesizing new DNA strands. Coordination is needed between synthesis of the leading and lagging strands at the replication fork.

1. Tanvi Lak hlani
22mbt019

2. • The two new DNA strands have
different modes of synthesis
• Replication requires a helicase and a
single strand binding protein
• Priming is required to start DNA
synthesis &coordination synthesis of
the lagging and leading strand 2
CONTENT

3. WHAT SHOULD YOU KNOW?
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• Leading Strand & Lagging Strand
• Primer
• Okazaki Fragments
• Helicase
• Single Strand Binding Protein

4. 4
SSB

5. 5
• As the replication fork advances, daughter strands must be
synthesized on both of the exposed parental single strands.
• The fork template strand moves in the direction from 5’-3’ on one
strand and in the direction from 3’-5’ on the other strand.
The Two New Dna strands Have Different Modes of
Synthesis
• DNA is synthesized only from a 5’ end towards a 3’ end on a
template that is 3’ to 5’. This problem is solved by synthesizing new
strand on the 5’ to 3’template in a series of short fragments, each
synthesized in “Backward” direction.
5’ 3’
3’ 5’
5’ 3’
3’ 5’

6. LEADING STRAND LAGGING STRAND
• On the forward strand DNA
synthesis can proceed
continuously in the 5’ to 3’
direction as the parental
duplex is unwound.
• On this strand a stretch of ss
parental DNA must be
exposed, and then a segment
is synthesized in the reverse
direction. Then these
fragments are joined together
to create an intact lagging
strand.
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7. 7
• Discontinuous replication can be
followed by the fate of very brief label of
radioactivity.
• The label enters newly synthesized DNA
in the form of short fragments of ~ 1000
to 2000 bases in length. These Okazaki
fragments are found in replicating DNA
in both prokaryotes and eukaryotes.
• After a long period of incubation, the
label enters larger segment of DNA,
Okazaki fragments are joined together
by covalent linkages.
• For a long time it was unclear whether
the leading strand synthesized in
discontinuous or continuous manner.

8. 8
• All newly synthesized DNA is found as a short fragments in E.coli.
Superficially this suggests that both strands are synthesized
discontinuously.
• But not all the fragment population represents Okazaki fragments;
some are pseudofragments that have been generated by a breakage
in a DNA stand that actually was synthesized as a continuous chain.
• The source of this breakage is the incorporation of some uracil into
DNA in place of thymine.
• When the uracil is removed by repair system, the leading strand has
breaks until a thymine is inserted.
• Thus, it is suggested that leading strand is synthesized continuously
while lagging strand is synthesized discontinuously. This is called
semi-discontinuous replication.

9. Replication Requires a Helicase and SSB Proteins
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• As replication fork advances, it unwinds the duplex DNA.
• One of the template strands is rapidly converted to duplex DNA as
the leading strand is synthesized.
• The other remains single stranded until a sufficient length has
been exposed to initiate synthesis of an Okazaki fragment
complementary to the lagging strand in backward direction.
• The generation and maintenance of ss DNA is therefore a crucial
aspect of replication.
• Teo types of function are needed to convert ds DNA to the ss DNA:
1. Helicase
2. Single Stranded Binding Protein

10. 10
HELICASE ENZYME
• A helicase is an enzyme that
separates the strands of DNA,
usually using the hydrolysis of ATP
to provide the necessary energy.
• It separates the strand of duplex
nucleic acid in a variety of
situation, ranging from stand
separation at the growing point of
a replication fork to catalyzing
migration of holiday junctions
along DNA.

11. 11
• There are 12 different helicase in E.coli.
• A helicase is generally multimeric.
• A common form of helicase is hexamer.
• This typically translocates along DNA by using its multimeric structure
to provide multiple binding sites.

12. 12
• In hexameric form, it is likely to have one
conformation that binds to duplex DNA and
another that binds to ss DNA.
• Alteration between them drives the motor that
melts the duplex and requires ATP hydrolysis
typically 1 ATP is hydrolyzed for each base pair
that is unwound.
• A helicase usually initiates unwinding at a single
stranded region adjacent to a duplex DNA.
• It may function with particular polarity, preferring
ss DNA with a 3’ end (3’-5’ helicase) or with a 5’
end (5’-3’end).
• Hexameric helicases typically encircle the DNA,
which allows them to unwind DNA processively
for many kilobases. This makes them ideally
suited as replicative DNA helicases.

13. 13
SINGLE STRANDED BINDING PROTEINS (SSB
Protein)
• A SSB proteins binds to a ss DNA, protecting it and
preventing it from reforming the duplex state. The SSB binds
typically in a cooperative manner in which the binding of
additional monomers to the existing complex is enhanced.
Eukaryotic SSB

14. 14
• The E.coli SSB is tetramer, eukaryotic
SSB (also known as RPA) is trimer.
• E.coli SSB is 74 kD that binds ss DNA
cooperatively.
• The significance of the cooperative
mode of binding is that the binding of
one protein makes it much easier for
another to bind.
• Thus, once the binding is started on a
particular DNA molecule, it is rapidly
extended until all of the ss DNA is
covered with the SSB protein.
Eukaryotic SSB

15. 15
• Under normal circumstances in vivo, the unwinding, coating and
replication reaction proceed together.
• The SSB binds to DNA as the replication fork advances, keeping
the two parental stands separate so that they are in the
appropriate condition to act as templates.
• SSB is needed in stoichiometric amounts at the replication fork
• It is required for more than one stage of replication; ssb mutants
have a quick stop phenotype, and are defective in repair and
recombination as well as in replication.

16. Priming Is Required to Start DNA Synthesis
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• A common feature of all DNA polymerases is that they cannot
initiates synthesis of a chain of DNA de novo, but can only
elongates a chain.
• The synthesis of new strand can only start from a preexisting
3’-OH end, and the template strand must be converted to a
single stranded condition.
• The 3’-OH end is called a primer.

17. 17
• Types of priming reaction:
• A sequence of RNA is synthesized on
the template, so that the free 3’-OH
end of the RNA chain is extended by
the DNA polymerase. This is
commonly used in replication of
cellular DNA and by some viruses.
• A performed RNA pairs with the
template, allowing its 3’-OH end to be
used to prime DNA synthesis. This
mechanism is used by retroviruses to
prime reverse transcription of RNA.

18. 18
• A primer terminus is generated
within duplex DNA. The most
common mechanism is the
introduction of a nick, as used
to initiate rolling circle
mechanism. In this case
preexisting strand is displaced
by new synthesis.
• A protein primase the reaction
directly by presenting a
nucleotide to the DNA
polymerase. This reaction is
used by certain viruses.

19. 19
• Priming activity is required to provide 3’-OH ends to start off the
DNA chains on both strands.
• The leading strand requires only one such initiation event, which
occurs at origin.
• There must be series of initiation events on the lagging strand,
because each Okazaki fragment requires its own start de novo.
Each Okazaki fragment start with primer sequence of RNA ~ 10
bases long that provides the 3’-OH end for extension by DNA
polymerase.
Leading strand
primer primer
Lagging strand
3’
5’ 3’
3’
5’
primer
5’ 3’
3’ 5’
3’
5’ 3'

20. 20
• A primase is required to catalyze the actual
priming reaction.
• In E.coli, this is provided by a special RNA
polymerase activity, the product of the dnaG
gene. The enzyme is single polypeptide of 60 kD.
• The primase is a RNA polymerase that is used
only under specific condition; that is, to
synthesize short stretches of RNA that are used
as primers for DNA synthesis.
• DnaG primase associates transiently with the
replication complex, and typically synthesizes a
~10 base primer.
• Primers start with the sequence pppAG
positioned opposite the sequence 3’ –GTC -5’ in
the template.

21. 21
• There are two types of priming reaction in E.coli:
• The oriC system, named for the bacterial origin, basically involves
the association of the DnaG primase with the protein complex at
the replication fork.
• The ΦΧ system, named originally for phage ΦX174, requires an
initiation complex consisting of additional components, called the
primosome. This system is used when damage causes the
replication fork to collapse and it must be restarted.

22. 22
• DnaB is the central components in both ΦX and
Oric replicons. It provides the 5’-3’ helicase
activity that unwinds DNA. Energy for the
reaction is provided by cleavage of ATP.
• Basically, DnaB is the active component required
to advance the replication fork.
• In oriC replicons, DnaB is initially loaded at the
origin as the part of large complex.
• It forms the growing point at which the DNA
strands are separated as the replication fork
advances.
• It is part of DNA polymerase complex and
interacts with the DnaG primase to initiate
synthesis of each Okazaki fragment on the
lagging strand.

23. Coordination Synthesis of the Lagging and
Leading Strands
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• Each new DNA strand, leading and
lagging, is synthesized by an
individual catalytic unit and behavior
of these two units is different
because the new DNA strands are
growing in opposite directions.
• One enzyme unit is moving in the
same direction as the unwinding
point of replication fork and
synthesizing the leading strand
continuously.
• The other unit is moving “backward”
relative to the DNA, along the
exposed single strand.

24. 24
• When synthesis of one
Okazaki fragment is complete,
synthesis of next Okazaki
fragment is required to start
new a new location
approximately in the vicinity of
the growing point for the
leading strand.
• This requires that DNA
polymerase III on the lagging
strand disengage from the
template, move to new
location, and be reconnected
to the template at a primer to
start a new Okazaki fragment .

25. 25
• E.coli , there is only a single DNA
polymerase catalytic subunit used in
replication, the DnaE polypeptide.
• Some bacteria and eukaryotic have
multiple replication DNA polymerases.
• In the Bacillus subtilis, there are two
different catalytic subunits. PolC is the
homolog to E.coli’s DnaE, is responsible
for synthesizing the leading strand.
• A related protein, DnaEBS is the catalytic
subunit that synthesizes the lagging strand.
• Eukaryotic DNA polymerases have same
general structure, with different enzyme
units synthesizing the leading and lagging
strand.

26. 26

27. References
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• Lewin’s Genes XI
• https://th.bing.com/th/id/R.711e1b56815aa51b16d04fa22d6259f9?rik=DWN
pHTI15KY6XA&riu=http%3a%2f%2fwww.mdpi.com%2fgenes%2fgenes-08-
00022%2farticle_deploy%2fhtml%2fimages%2fgenes-08-00022-
g002.png&ehk=0YlKTpnWltwXb410T5aD2MmPgiasYomI3ut1dFYroD8%3d
&risl=&pid=ImgRaw&r=0
• https://d20khd7ddkh5ls.cloudfront.net/dna_replication_eukaryotic.jpg
• https://image2.slideserve.com/5131236/single-strand-dna-binding-ssb-
protein-l.jpg
• https://www.researchgate.net/profile/Change_Tan/publication/313744699/fi
gure/download/fig4/AS:462060455763972@1487175206076/A-
comparison-of-DNA-replication-initiation-in-bacteria-E-coli-and-bakers-
yeast-S.png

28. Thank you
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