1) DNA replication begins with the unwinding of the DNA double helix at an origin of replication site. 2) This forms a replication fork with leading and lagging strands that are copied semi-conservatively to produce two identical copies of DNA. 3) RNA primers, DNA polymerases, helicase and single-strand binding proteins work together to separate the strands and synthesize new DNA in the 5’-3’ direction along the template.

1. Replication/steps
in prokaryotic DNA
synthesis

2. Introduction
When the two strands of the DNA doble
helix are seperated each can serve as a
template for replication of a new
complementary strand. This produces two
daughter molecules, each of which
contain two DNA strands with an
antiparallel direction. This process is
called semiconservative replication.

3. Why semiconservative
Because the parental duplex is separated
into two halves and therefore it is not
conserved as entity, each of the individual
parental strands remain intact in one of
the two new duplexes.
The enzyme involve in the DNA replication
process are template-directed polymerase
that can synthesis complementary DNA
strand with extraodinary fidelity.

4. Requirements for DNA
synthesis
1) Activated deoxy nucleoside triphosphate
e.g. dATP, dGTP, dCTP, dTTP
2) DNA template
3) DNA polymerase
4) Primer (RNA primer)
5) DNA polymerase associated protein and
enzyme- primase, helicase, SSBP,
Topoisomerase I &II , DNA ligase
6) Magnesium ion

5. Steps of replication
1) Initiation
2) Elongation
3) Termination
4) Synthesis of new histone
5) Reconstitution of chromatin structure with
histone

6. Criteria of replication
• Semiconservative process
• Symmetric process
• Nonselective process
• Need primer
• Template is copied from 3´-5´ direction
• High fidelity with erros rate <10-18
per base
pair
• No post replication modification
• Bidirectional and semidiscontinuous
process

7. Separation of DNA strand
The parental double helical DNA to be
replicated they must first separate or melt
over a small region because the
polymerase use only ssDNA for
replication.
The replication begins at a single, unique
nucleotide sequence a site called origin of
replication- that referred as consensus
sequence.

8. Separation of DNA strand
The order of nucleotides is essentially the
same at each site. The site include a
short sequence composed almost
exclusively of AT base pair that facilates
melting.
In eukaryotes, replication begins at multiple
site along the DNA helix.

9. Formation of replication fork
As the two strands unwind and separate
they form a V where active synthesis
occurs. The region is called the replication
fork. Replication of dsDNA is bidirectional-
that is replication move in opposite
directions from the origin generating
replication bubble.

10. Formation of replication fork
Proteins required for DNA strand separation:
Initiation of DNA replication requires the
recognition of the origin of the replication
by a group of protein that form the
prepriming complex- that maintain and
stabilize separation and for unwind the
double helix ahead the replication fork

11. Formation of replication fork
1)protein: DnaA protein binds with specific
nucleotide sequence at the origin of
replication, melting is ATP dependent &
results in strand seperation, with formation
of localized regions of ssDNA.
2) DNA helicases: these enzyme bind to
ssDNA near the replication fork and then
move into neighboring double strand
region, forcing the strand apart.

12. Formation of replication fork
3)SSB protein:these protein bind to ssDNA
produced by helicases. The protein keep
the two strand of the DNA separated in
the area of the replication origin thus
providing the single strand template for
polymerase. Also protect the DNA from
nucleases that degrade.

13. Formation of replication fork
• Solving the problem of supercoils:
If the twisted strands are uncoiled then
there is a problem of supercoil. If the cord
is twisted in the direction of tightening
coils, the cord will wrap around itself-
positive supercoils and if the cord is
twisted in the direction of loosening
direction, the cord will wrap around itself-
negative supercoil

14. Formation of replication fork
DNA tropoisomerase which is responsible
for removing supercoils in the helix. There
are two types of tropoisomerases-
1)Type I DNA topoisomerases: don’t require
ATP and relax the negative supercoils.
2)Type II DNA topoisomerase: ATP
dependent process and relax both positive
and negative supercoils.

15. Direction of DNA replication
The DNA polymerase responsible for
copying DNA templates in 3´-5´direction
and they synthesize the new template only
in 5 ´-3 ´ direction.
1)Leading strand: the strand copied in the
direction the replication fork and it is
synthesized continuously.

16. Direction of DNA replication
2) Lagging strand: the strand copied away
from replication fork and synthesized
discontinuously with small fragments of
DNA being copied near the replication
fork. These short stretches of
discontinuously DNA termed as Okazaki
fragments are eventually joined to become
a single continuous fragment. The DNA
produced by these mechanism is termed
lagging strand.

17. RNA primer
DNA polymerase can’t initiate synthesis of
complementary strand of DNA on a totally
single stranded template. So they require
a primer that is a RNA primer- is a short,
double stranded region consisting of RNA
base paired to the DNA template with a
free hydroxyl group on the 3´-end of the
RNA strand. This hydroxyl group serve as the
first acceptor of deoxynucleotide by action of
DNA polymerase.

18. RNA primer
1. primase- a specific RNA polymerase
called primase synthesized the short
stretches of RNA that is approximately
ten nucleotides long that is
complementary and antiparallel to the
DNA template. These short RNA
sequence are constantly being
synthesized at the replication fork on the
lagging strand, but one RNA sequence in
leading strand.
2. primosome

19. Chain elongation
• Prokaryotic and eukaryotic DNA
polymerase elongate a new DNA strand
by adding deoxyribonucleotide one at a
time to the 3 ´ end of the growing chain.
The sequence of incoming nucleotide are
dictated by template strand.
1. DNA polymerase III- DNA chain
elongation is catalyzed by DNA
polymerase III.

20. • Using 3´hydroxyl group of the RNA primer
as well as acceptor of the first
deoxynucleotide. So DNA polymerase
binds the incoming nucleotide to single
strand DNA template. DNA polymerase III
is a highly processive enzyme that remain
bound to the template strand and moves
along and does not diffuse away. The
processivity of DNA polymerase is the
result of β subunit that form a ring
encircling the DNA. The DNA strand
grows along 5´-3´ direction.

21. 2. Proofreading of newly synthesized DNA-
it is highly for survival of an organism that
DNA replication process should be with
few errors as possible. Misreading of
template is dangerous perhaps lethal
mutation. To ensure fidelity DNA
polymerase III has an addition 3´-5´
exonuclease activity, each nucleotide is
added to chain is correctly matched to its
complementary bases. If there is ant
mismatched nucleotide its is removed
hyderlytically.

22. Excision of RNA primers and
their replacement by DNA
• Polymerase III continue to synthesized
DNA on the lagging strand until it is
blocked by proximity to an RNA primer.
RNA primer is excised and the gap is filled
by DNA polymerase I.
1. 5´-3´ exonuclease activity: in addition
having 5´-3´ polymerase activity that
synthesized DNA and 3´-5´ exonuclease
activity that proofreads the newly
synthesized DNA chain like DNA

23. chain like DNA polymerase III, DNA
polymerase I also have 5´-3´ exonuclease
activity.
What is exonuclease activity:exonuclease
activity because they remove one
nucleotide at a time from the end of the
DNA chain rather than cleaving the chain
internally that done by endonuclease.
How the RNA primers are removed – DNA
polymerase I locates the space between
the 3´-end of newly synthesized DNA by

24. DNA polymerase III and the 5´ end of the
adjacent RNA primer. Then DNA
polymerase I hydrolytically removes the
RNA nucleotide ahead in 5´-3´ direction
and DNA polymerase I synthesis the new
DNA in 5´-3´ direction and proofreading
also done by 3 ´-5 ´ direction.
Difference between 5 ´-3 ´ and 3 ´-5 ´
exonuclease activity:

25. • 5´-3´ exonuclease activity by DNA
polymerase I and 3´-5´ by DNA
polymerasre I & III.
• 5´-3´ exonuclease remove one nicleotide
at a time from a region of properly base
paired.
• 5´-3´ exonuclease can remove one to ten
nucleotide at a time.

26. DNA ligase
• The final phosphodiester linkage between
5´ phosphate group on the DNA chain
synthesized by DNA ploymerase III & 3´
hydroxyl group on the chain made by DNA
polymerase I is catalyzed by DNA ligase.