DNA replication In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritance.

1. DNA REPLICATION

3. Flow of genetic information --- the “Central
Dogma”

4. Cell Cycle
• A typical cell goes througha
process of growth,
development, and reproduction
called the cell cycle.
• Most of the cycle is called
interphase.
INTERPHAS
E

5. • Eukaryotes have a network of regulatory
proteins, known as the cell cycle control
system to monitors the cell cycle.
• The checkpoints regulated by a family of
protein kinases known as the cyclin-
dependent kinase (CDKs).
• That bind to different classes of regulator
proteins known as cyclines.
• During early G1, the transcriptional
repressors Rb (retinoblastoma), p107 and
p130, known as pocket proteins, bind to the
E2F transcription factors to prevent G1-to-S

7. Replication
Replication- the synthesis of DNA using itself
as a template.
Synthesis of new DNA from existing DNA
template.
Must take place prior to cell division.

8. Initiation
•This process is initiated at particular points in
the DNA, known as "origins", which are
targeted by initiator proteins.
•Sequences used by initiator proteins tend to
be "AT-rich" (rich in adenine and thymine
bases)

9. Elongation
•DNA polymerase has 5'-3' activity. All known DNA replication
systems require a free 3' hydroxyl group before synthesis can be
initiated.
•All cellular life forms and many
DNA viruses, phages and plasmids use a primase to synthesize a short
RNA primer with a free 3' OH
•The retroelements (including retroviruses) employ a transfer RNA tha
primes DNA replication by providing a free 3′ OH that is used for
elongation by the reverse transcriptase

11. DNA-pol of eukaryotes
DNA-pol : elongation DNA-pol III
DNA-pol : initiate replication and
synthesize primers
DnaG,
primase
DNA-pol : replication with low fidelity
DNA-pol : polymerization in
mitochondria
DNA-pol : proofreading and filling gap DNA-pol I
repairing
11

12. Termination
• Termination at a specific locus, when it
occurs, involves the interaction between
two components: (1) a termination site
sequence in the DNA, and (2) a protein
which binds to this sequence to physically
stop DNA replication

13. • Eukaryotes initiate DNA replication at
multiple points so replication forks meet
and terminate at many points .
• But DNA replication is unable to reach the
very end of the chromosomes.
• Due to this problem, DNA is lost each
replication cycle from the end of the
chromosome.

14. • Telemores are regions of repetitive DNA in
the ends
• Shortening of the telomeres is a normal
process in somatic cell
• As a result, cells can only divide a certain
number (Hayflick limit .) Within the germ
cell line, telemorase extends the repetitive
sequences of the telomere to prevent
degradation. Telomerase can become
mistakenly active in somatic cells,
sometimes leading to Cancer

16. Replication requires the coordinated regulation of many enzymes and
processes
• 1/ Recognition of origin (where helicase is starting to unwind DNA).
• 2/Unwinding of DNA strands by Helicase.
• 3/ ssDNA Binding Protein will attach to each strand to prevent rewinding.
• 4/Synthesis of RNA primer complementary to DNA by Primase.
• 5/Formation of new DNA strand in 5`-3` Direction by DNA polymerase.
• 6/3’to 5’ exonuclease activity proofreads and repair mistakes
• 7/ In Lagging strand addition of RNA primer by Primase.
• 8/ Okazaki fragments
• 9/ Excision of RNA primers by exonuclease enzyme.
• 10/ Fusion of Okazaki fragments by Ligase enzyme.
• 11/ Topoisomerase relieves supercoiling and then resealed nicks

17. In S phase, DNA replication begins at origins of replication
that are spread out across the chromosome
An origin of replication (replicon) is a site in a DNA molecule at
which helicase unwinds the double helix.
A prokaryote chromosome has only one origin of replication as
eukaryotic cells have many sites known as autonomous replicating
sites (ARS).

18. Enzymes in DNA replication
Helicase unwinds
parental double helix
Binding proteins
stabilize separate
strands
DNA polymerase
binds nucleotides
to form new strands
Ligase joins Okazaki
fragments and seals
other nicks in sugar-
phosphate backbone
Primase adds
short primer
to template strand
Exonuclease removes
RNA primer and inserts
the correct bases
Primase
Primer

19. Binding proteins prevent single strands from rewinding.
Replication
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
5’
3’
5’
3’
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.
3’
5’
5’
3’

20. Replication
Overall direction
of replication
5’
3’
5’
3’
5’
3’
3’
5’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.

21. Replication
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
5’
5’
Overall direction
of replication
5’
3’
5’
3’
3’
3’
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.
Helicase

22. Replication
5’
5’
3’
5’
3’
3’
5’
3’
Overall direction
of replication
Leading strand synthesis continues in a
5’ to 3’ direction.

23. Replication
3’
5’ 5’
5’
3’
5’
3’
3’
5’
3’
Overall direction
of replication
Okazaki fragment
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

24. 5’
Replication
5’
5’
3’
5’
3’
3’
5’
3’
Overall direction
of replication
3’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Okazaki fragment

25. Replication
5’
5’ 3’
5’
3’
3’
5’
3’
3’
5’ 5’
3’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

26. Replication
3’
5’
3’
5’
5’ 3’
5’
3’
3’
5’ 5’
3’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

27. Replication
5’
5’
3’ 3’
5’
3’
5’ 3’
5’
3’
3’
5’
Exonuclease enzymes remove RNA primers.

28. Replication
Exonuclease enzymes remove RNA primers
and replace it with DNA nucleotides
Ligase forms bonds between sugar-phosphate
backbone.
3’
5’
3’
5’ 3’
5’
3’
3’
5’

29. Replication
5’
3’
5’
3’
5’
3’
3’
5’
5’
5’
3’
5’
3’ 3’
5’
3’
5’
3’
5’
3’
3’
5’
5’
3’
3’
5’ 5’
5’
3’
5’
3’ 3’
5’
3’
5’
5’ 3’
5’
3’
3’
5’ 5’
3’
5’
3’
3’
5’
3’
5’ 3’
5’
3’
3’
5’
3’
5’
3’
5’ 3’
5’
3’
3’
5’

30. 30

31. • Also called -protein in prokaryotes.
• It cuts a phosphoester bond on one DNA strand,
rotates the broken DNA freely around the other
strand to relax the constraint, and reseals the
cut.
Topoisomerase I (topo I)
31

32. • It is named gyrase in prokaryotes.
• It cuts phosphoester bonds on both strands of
dsDNA, releases the supercoil constraint,
and reforms the phosphoester bonds.
• It can change dsDNA into the negative
supercoil state with consumption of ATP.
Topoisomerase II (topo II)
32

35. Fidelity of replication
• DNA replication has a very high degree of fidelity as the
genetic complement of the resultant daughter cells must be
the same as the parental cell.
• Accuracy of DNA polymerases is essential.
• Error rate is less than 1 in 108
• Due in part to “reading” of complementary bases
• also contains its own proofreading activity

38. • Replication errors can also involve
insertions or deletions of nucleotide bases
that occur during a process called strand
slippage.

39. When DNA damage occurs
• when the cell detects any defects which necessitate
it to delay or halt the cell cycle in G1,
• The rapid response involves phosphorylation events
that initiate with either kinase ATM or ATR ,
which act as sensors,
• These kinases phosphorylate and activate the
effector kinases Chk2 and Chk1, respectively
• which in turn phosphorylate the phosphatase
Cdc25A, thus marking it for ubiquitination and
degradation

40. • To maintain the arrest, another response is
initiated, by which Chk2 or Chk1
phosphorylate p53, a tumor suppressor, and
this stabilizes p53 by preventing it from
binding Mdm2

41. Cancer
• DNA repair processes and cell cycle
checkpoints have been intimately linked
with cancer .
• The loss of ATM has been shown to
precede lymphoma developmen.
• Disruption of Chk1 in mice led significant
misregulation of cell cycle checkpoints, an
accumulation of DNA damage, and an
increased incidence of tumorigenesis.

42. • P53
• Rb
• BRCA1
• BRCA2

43. Antibiotic affecting DNA
Replication
• DNA Gyrase (Topoisomerase) is present in prokaryotes
and some eukaryotes, but the enzymes are not entirely
similar in structure or sequence, and have different
affinities for different molecules. It is not present in
humans. This makes gyrase a good target for antibiotics.
Two classes of antibiotics that inhibit gyrase are:
• The aminocoumarins (including novobiocin).
Aminocoumarins work by competitive inhibition of energy
transduction of DNA gyrase by binding to the ATPase
active site located on the GyrB subunit.

44. • The quinolones (including Nalidixic
acid and Ciprofloxacin). Quinolones bind these
enzymes and prevent them from decatenating
replicating DNA. Quinolone-resistant bacteria
frequently harbor mutated topoisomerases that resist
quinolone binding.
• DNA gyrase has two subunits,which in turn have two
subunits each, i.e. 2A and 2B SUBUNITS.The A
subunit carries out nicking of DNA,B subunit
introduces negative supercoils,and then A subunit
reseals the strands.Fluorquinolones bind to the A
subunit and interfere with its strand cutting and