ABOUT THE REPLICATION FORK IN BOTH PROKARYOTES AND EUKARYOTES

1. REPLICATION
FORK
BY
KANCHANA.A.S
I MSc BIOCHEMISTRY

2. Replication fork in prokaryotes:
The first step in DNA replication is the separation of the two
DNA strands that make up the helix that is to be copied. DNA
Helicase untwists the helix at locations called replication origins.
The replication origin forms a Y shape, and is called a replication
fork.
•Replication fork is the structure which is formed when DNA HELICASE
( DNA B PROTEIN )Get attaches to the ORI C Region of bacterial
chromosome.
•The function of DNA HELICASE Is to break the hydrogen bond between
base pairs and thereby unwind the DNA Strand

3. •This unwinding of the DNA strand causes positive super coiling
ahead of each replication fork
•An enzyme known as Topoisomerase II( also known as dna gyrase),
will travel in front of the DNA Helicase and prevents positive super
coiling.

5. What prevents the dna strands from coming back together ?
•Dna replication requires SINGLE STRANDED BINDING
PROTEINS (SSBP)
i. This will get binds to the parental dna strand and thereby it will
prevent them from reforming a double helix.
i. In this way , the bases within the parental strands are
kept in an exposed condition that enables them to hydrogen
bond with individual nucleotides.

6. THE EFFECT OF SINGLE-STRAND DNA-BINDING PR
OTEINS (SSB PROTEINS) ON THE STRUCTURE OF SI
NGLE-STRANDED DNA.

7. •As a result two strands are formed :
A. Leading strand
B. Lagging strand
RNA PRIMING:
•The next event in dna replication involves the synthesis of short strands
of RNA called RNA primer
•These strands of RNA are synthesized by the linkage of ribonucleotides
via an enzyme known as primase.
•This enzyme will produce short strands of RNA
( 10 – 12 nucleotides).
a. In leading strand only one primer is made at the ori
b. In lagging strand multiple primers are made

8. RNA primer synthesis. A schematic view of the reaction catalyzed by DNA
primase, the enzyme that synthesizes the short RNA primers made on the
lagging strand using DNA as a template.

9. •Next enzyme which comes into action is the
dna polymerase
•This enzyme is responsible for synthesising the dna of the leading
and the lagging strand
But this dna polymerase enzyme has two drawbacks :
•This enzyme lacks the ability to initiate new strand of
nucleic acid .
•This can only elongate pre existing strand starting with an RNA
primer.
•This will attach the nucleotides only in 5’ – 3’ direction
This is the reason why leading and the lagging strands are
synthesised in different ways.

10. SYNTHESIS OF LEADING STRAND
•Only one rna primer is made at the origin
•Dna polymerase enzyme will attach nucleotides in 5’-3’ direction as it slides
towards the opening of the replication fork .
•The synthesis of leading strand is continuous
•The incoming nucleotide is added to the hydroxyl group at 3’ end of growing
chain
•Precursor for dna synthesis : deoxyribonucleotide 5’ triphosphate .
•Proceeding from deoxyribose outwards 3 groups are designated
I. Alpha phosphate group
II. Beta phosphate group
III. Gamma phosphate group

11. •Upon polymerisation , high energy bond between alpha and
beta phosphate group is cleaved
•Released as a molecule of pyrophosphate.
•New bond is made between innermost of incoming nucleoti
de and 3’OH of previous nucleotide at end of growing chain
Thereby the dna polymerase enzymes elongates the DNA s
trand and new daughter dna is synthesised.

12. SYNTHESIS OF LAGGING STRAND
 Requires many rna primers
 The synthesis of dna also elongates in a 5' to 3' direction but happens
away from the replication fork.
In the lagging strand , rna primers must repeatedly initiate the synthesis of
short segments of dna .
 Thus the synthesis has to be discontinuous
 Length of these fragments is 1000-2000 nucleotides in bacteria.
 Each fragment contains a short rna primer at the 5’end which is made by
primase
 These small fragments are called as okazaki fragments.

13.  To complete the synthesis of okazaki fragments three events a has to occur :
I. Removal of rna primer ( by dna pol I)
II. Dna synthesis in area where rna primer has been removed
III. Covalent attachment of nucleotides.
• DNA polymerase I would remove the rna primer from the first okazaki fragment and
then synthesise dna in the vacant region by attaching the nucleotide to the 3’ end of the
second okazaki fragment .
• The Okazaki fragments were shown to be polymerized only in the 5′-to-3′chain
direction and to be joined together after their synthesis to create long DNA chains.
After the gap has been completely filled in a covalent bond is still missing between the
two adjacent nucleotides and this filled by dna ligase ( joins the adjacent nucleotide by
forming a covalent bond between them ).
• Dna ligase in bacteria requires NAD+
That ends the synthesis of dna in replication fork.

14. The reaction catalyzed by DNA ligase:

15. TERMINATION :
•Opposite to ori c is two termination sequence
•Contain protein called tus protein (termination utilization substances )
•Dna replication ends when oppositely advancing forks meet ( since it is
a circular chromosome)
•Finally dna ligase covalently links the two daughter strand creating two
circular double stranded dna .

16. REPLICATION IN PROKARYOTES

17. REPLICATION FORK (eukaryotes)
Here dna replication begins at the origin by binding of orc to ori
The binding of mcm(mini chromosome maintenance ) Helicase
at the origin completes a process called dna replication licensing
( only those origins with mcm Helicase can initiate the dna
synthesis)
The mcm complex along with some additional proteins will
assemble two divergent replication forks at each replication origin
Bidirectional
Multiple origin spaced 30 – 300 kb apart
DNA sequence between 2 origin of replication - Replicon

19. LEADIND STRAND SYNTHESIS:
•During DNA replication, the replisome will unwind the parental duplex
DNA into a two single-stranded DNA template replication fork in a 5' to 3‘
direction.
• The leading strand is the template strand that is being replicated in the
same direction as the movement of the replication fork.
•This allows the newly synthesized strand complementary to the original
strand to be synthesized 5' to 3' in the same direction as the movement of
the replication fork.
•Once an RNA primer has been added by a primase to the 3' end of the
leading strand, DNA synthesis will continue in a 3' to 5' direction
• DNA Polymerase ε will continuously add nucleotides to the template
strand

20. •To achieve the high degree of processivity required for DNA replication, DNA
polymerases associate with ring-shaped sliding clamps(PCNA) that encircle the
template DNA and slide freely along it
•specifically targets them to sites where DNA synthesis is initiated and orients them
correctly for replication.
•Such a method is performed by multisubunit complexes known as clamp loaders,
(REPLICATION FACTOR C) which use ATP to open sliding clamp rings and place
them around the 3′ end of primer–template (PT) junctions.

21. PCNA(proliferating cell nuclear antigen)
Multimeric protein
•Acts as clamp – to keep DNA pol
•From dissociating off leading strand
•Increases the activity of DNA pol epsilon
•PCNA is often viewed as a regulatory cofactor for DNA polymerases
REPLICATION FACTOR C( RFC)
Clamp loader or match marker
Assist in DNA pol delta to form clamp between DNA
and PCNA
Set up link between dna pol alpha and dna pol delta
Leading strand and lagging strand synthesis can take
place simultaneously

22. CLAMP AND CLAMP LOADER

23. LAGGING STRAND SYNTHESIS
•Lagging strand synthesis of okazaki fragments initiated same way as leading strand
synthesis
•An okazaki fragment consist of 150-200 nucleotides
•Rna primer is synthesized by dna pol alpha due to its primase activity
•Primer is then extended by dna pol delta due to its 5’-3’ activity
•Here the rna primer is removed by an enzyme called flap endonucleases ( fen i)
•Flap endonucleases gets it name because it removes small pieces of rna flaps that rae ge
nerated by the action of dna pol delta
•For the removal of long flaps dna 2 Helicase or nuclease enzyme will help to cut the
long flaps into smaller ones and then these are removed by fen 1 As they remove the gap
is filled by dna pol epsilon by its 5’-3’ activity
•Dna ligase joins the okazaki fragments.

24. The synthesis of one of the many DNA fragments on the
lagging strand

25. TERMINATION:
•When the replication forks meet each other termination occurs result in the formation
of two duplex dna
•Even though replication terminated At the 5’end of telomeric part of
The newly synthesised dna is found to have shorter dna strand than parental strand
•This shortage is corrected by telomerase and then only the actual replication is compl
eted

26. RECENT FINDING:

27. REFERENCES:
1. GENETICS ANALYSIS AND PRINCIPLES BY BROOKER
2. MOLECULAR BIOLOGY OF GENE BY JAMES D WATSON ,
TANIA A BAKER ,STEPHEN P BILL,ALEXANDER GRAM )
3. ESSENTIAL CELL BIOLOGY BY ALBERTS,BRAY,JOHNSON
ET AL
4. THE CELL, A MOLECULAR APPROACH BY COOPER
5. TEXTBOOK OF MOLECULAR BIOLOGY BY M.PRAKASH
6. MOLECULAR BIOLOGY BY E. TROOP

28. THANK YOU