1. DNA replication is the process where parental DNA is used as a template to produce identical copies of DNA or daughter DNA. It ensures faithful transmission of genetic material to offspring. 2. Replication starts at specific origins of replication and involves initiation, elongation, and termination phases. Enzymes involved include DNA polymerases, helicases, primases, ligases and more. 3. Eukaryotic replication is more complex, with multiple polymerases and regulated initiation. Telomerase is required for end-replication and chromosome integrity. 4. DNA repair mechanisms include base excision, nucleotide excision, mismatch and double-strand break repair to fix errors and damage via pathways like non-homologous

1. S.RAJASEKHAR
TAM/16-43
BIOTECHOLOGY

2. DNA replication
• A process in which daughter DNAs are
synthesized using the parental DNAs as the
template.
• Transferring the genetic information to the
descendant generation with a high fidelity.
2
replication
parental DNA
daughter DNA

3. DNA Replication Process

4. • The replication starts at a particular point called
origin of replication (ori).
• The origin of E. coli, ori C, is at the location of 82.
• The structure of the origin is 248 bp long and AT-
rich.
Replication of prokaryotes
a. Initiation:
4

5. Formation of preprimosome
5

6. • DnaA recognizes ori C.
• DnaB and DnaC join the DNA-DnaA complex,
open the local AT-rich region, and move on the
template downstream further to separate
enough space.
• DnaA is replaced gradually.
• SSB protein binds the complex to stabilize ssDNA.
Formation of replication fork
6

7. Unwinding of DNA

8. • The supercoil constraints are generated ahead of
the replication forks.
• Topoisomerase binds to the dsDNA region just
before the replication forks to release the
supercoil constraint.
• The negatively supercoiled DNA serves as a
better template than the positively supercoiled
DNA.
Releasing supercoil constraint
8

9. 9

10. 10

11. • Primase joins and forms a complex called
primosome.
• Primase starts the synthesis of primers on the
ssDNA template using NTP as the substrates in the
5´- 3´ direction at the expense of ATP.
• The short RNA fragments provide free 3´-OH
groups for DNA elongation.
Primer synthesis
11

12. Dna A
Dna B
Dna C
DNA topomerase
5'
3'
3'
5'
primase
Primosome complex
12

13. • dNTPs are continuously added to the primer or
the nascent DNA chain by DNA-pol III.
• The nature of the chain elongation is the series
formation of the phosphodiester bonds.
• The core enzyme catalyze the synthesis of
leading and lagging strands, respectively.
b. Elongation
13

14. 14

15. • The synthesis direction
of the leading strand
is the same as that of
the replication fork.
• The synthesis direction
of the latest Okazaki
fragment is also the
same as that of the
replication fork.
15

16. • Primers between Okazaki fragments are digested by
DNA pol I.
• The gaps are filled by DNA-pol I in the 5´→3´direction.
• The nick between the 5´end of one fragment and the 3
´end of the next fragment is sealed by ligase.
Lagging strand
16

17. 3'
5'
5'
3'
RNAase
POH
3'
5'
5'
3'
DNA polymerase
P
3'
5'
5'
3'
dNTP
DNA ligase
3'
5'
5'
3'
ATP
17

18. A total of 5 different DNAPs have been reported in E. coli
• DNAP I: functions in repair and replication .
• DNAP II: functions in DNA repair .
• DNAP III: principal DNA replication enzyme.
• DNAP IV: functions in DNA repair
• DNAP V: functions in DNA repair.
To date, a total of 14 different DNA polymerases have been
reported in eukaryotes.

19. Termination of replication
In prokaryotes:
DNA replication terminates when replication
forks reach specific “termination sites”.
• the two replication forks meet each other on
the opposite end of the parental circular DNA .

20. Termination of
Replication
• Occurs at specific site
opposite ori c
• termination sequences:
ter sequences~350 kb
Tus Protein-arrests
replication fork
motion

21. • Flanked by 6 nearly identical non-
palindromic*, 23 bp terminator (ter) sites.
• termination utilization substance (Tus) binds
to the ter sequences and stops the movement
of the replication forks.

22. Replication Fork

23. DNA Replication in Eukaryotes

24. Complex Process
• DNA replication in eukaryotes divided into
three stages
• 1.Initiation: licensing and activation. (The origin binding
proteins (OBP), form origin recognition complex (ORC), are
required for assembly of the pre-RC formation of pre-
replication complex (pre-RC) -> initiation of replication
complex (RC)
• 2.Elongation
• 3.Termination: telomere and telomerase

25. Initiation of Replication
• It is the first step in eukaryotic replication
in which most of the proteins combines to
form Pre – Replicative complex (Pre-RC).
• Involved proteins
Origin Recognition complex (ORC)
Cell division cycle 6( Cdc 6)
Chromatin licensing and DNA Replication
factor 1( Cdt 1)
Minichromosome Maintenance Protein
Complex (Mcm 2-7)

26. Steps in initiation

28. • The activity of Cdt 1 during the cell cycle is
regulated by a protein called Geminin.
• It also inhibits Cdt 1 activity during the S
phase in order to prevent the re-replication
of DNA, Ubiquitination and proteolysis.

29. Functions of Mcm Complex
• Minichromosome Maintenance Complex has
helicase activity and inactivation of any of the
six protein will prevent the progress of
formation of replication fork.
• It also has ATPase activity. A mutation at any
one of the Mcm protein complex will reduce
conserved ATP binding site.
• Mcm complex is a hexamer with Mcm2, Mcm
3, Mcm 4,Mcm 5, Mcm 6, Mcm 7.

30. origins of replication (Oris)
recognized by the origin recognition complex (ORC) of proteins
Cdt1 and Cdc6 (Cdc18 in fission yeast) recruit Mcm2-7at replication
origins
prereplication complex (pre-RC)
Licensed
Fired
multiple phosphorylation events carried out by cyclin E-CDK2
origin melting occurs and DNA unwinding by the helicase generates
ssDNA, exposing a template for replication
The replisome then begins to form

31. Elongation
• Once the complex forms and cell pass into S phase,
then unwinding of DNA strand takes place.
• Unwinding takes place by enzyme Helicases and it
leads of exposure of 2 DNA templates.
• After unwinding, polymerization of the daughter strands
takes place.
• It occurs with help of DNA polymerase enzymes.

32. • There are total 14 DNA polymerase enzymes
indentified till now but only 3 are involved in
replication process.
• They are :
1.DNA polymerase alpha
2.DNA polymerase epsilon
3.DNA polymerase delta

33. • DNA polymerase alpha :
• DNA polymerase αassociated with enzyme Primase, forms RNA primer
which are 8-10 nucleotide long.
• Later DNA polymerase α elongates this RNA primer 10 to 20 DNA bases
and then leaves the place.
• Elongation take place in 5’ to 3’ direction.
• DNA polymerase ε :
• DNA polymerase ε synthesis nucleotides on the leading strand.
• It will continuously add nucleotides leading to continous process of
replication.
• Thus it will require only one RNA primer at the beginning.

34. Polymerase switch
δ
ε

36. DNA polymerase δ :
•DNA polymerase δ helps the synthesis of DNA on lagging strand.
•On the lagging strand multiple RNA primers are required.
•On the lagging strand, DNA polymerase δ synthesize small fragments
of DNA called Okazaki fragments.
•At the end of each Okazaki fragment, DNA polymerase δ runs to
previous Okazaki fragment and replaces the RNA primer nucleotides
with Dna nucleotides.
•this leads to flap formation which is removed and the nick between is
replaced by enzyme DNA ligases .
•This process is known as Okazaki fragments maturation.

37. Polymerase Location
Size of
catalytic
subunit (kD)
Biological
function
Alpha (α)/primase nucleus 160-185
Priming &
Lagging strand
replication
Delta (δ) nucleus 125
Lagging strand
replication
Epsilon (ε) nucleus
210-230 or 125-
140
Leading strand
replication &
DNA repair
Beta (β) nucleus 40 DNA repair
Gamma (γ) mitochondria 125
Mitochondrial
DNA replication
DNA polymerase of eukaryotic cellsHigh fidelity enzyme

38. Polymerase Location Biological function
Zeta (ζ) nucleus Thymine dimer bypass
Eta (η) nucleus Base damage repair
Iota (ι) nucleus Required in meiosis
Kappa (κ) nucleus
Deletion & base
substitution
DNA polymerase of eukaryotic cells
Low fidelity enzymes

39. Removal of
RNA primers
by
FLAP
endonucleas
e

40. 3'
5'
5'
3'
3'
5'
5'
3'
connection of discontinuous
3'
5'
5'
3'
3'
5'
5'
3'
segment
c. Termination
40

41. Termination
• The termination of replication in eukaryotic cells occures by
telomere regions and telomerase. Telomeres extend the 3' end of
the parental chromosome beyond the 5' end of the daughter
strand.
• The RNA component of telomerase anneals to the single stranded
3' end of the template DNA and contains 1.5 copies of the
telomeric sequence.
• Telomerase contains a protein subSunit that is a reverse
transcriptase called telomerase reverse transcriptase or TERT.
TERT synthesizes DNA until the end of the template telomerase
RNA and then disengages.

43. Step 1 = Binding
Step 3 = Translocation
The binding-
polymerization-
translocation cycle can
occurs many times
This greatly lengthens
one of the strands
The complementary
strand is made by primase,
DNA polymerase and ligase
RNA primer
Step 2 = Polymerization

44. • The eukaryotic cells use telomerase to
maintain the integrity of DNA telomere.
• The telomerase is composed of
telomerase RNA
telomerase association protein
telomerase reverse transcriptase
• It is able to synthesize DNA using RNA as the
template.
Telomerase
44

45. 45

46. • Telomerase may play important roles is
cancer cell biology and in cell aging.
Significance of Telomerase
46

47. DNA REPAIR MECHANISMS

48. Single-strand damage
•Base excision repair (BER)
•Nucleotide excision repair (NER)
•Mismatch repair
Double-strand breaks
•Non-homologous end joining (NHEJ)
•Homologous recombination (HR)

49. Double-strand break repair -- Homologous
recombination pathways
•RecBCD recognizes
ends and unwinds and
degrades DNA until it
encounters a chi site.
•Nuclease activity is
suppressed on that
strand, generating a
ssDNA 3’ overhanging
end that initiates
recombination.
•RecA mediates strand
exchange

50. • RecA binds preferentially to SS DNA and will
catalyze invasion of a DS DNA molecule by a
SS homologue.
• One of the strands in the duplex is transferred
to the single strand.
• The other strand of the duplex is displaced.

51. Holiday JunctionHoliday Junction

52. Holiday Junction cleavageHoliday Junction cleavage
ResolutionResolution
patch
recombinan
t
splice
recombinan
t

54. Initiation of HR at the Molecular LevelInitiation of HR at the Molecular Level
Spo11: The Catalytic Activity for
Double-Strand Break Formation
DSB formation requires
additional proteins whose
activities are not yet
characterized.

55.  Recombinase as RecA/
Rad51/ Dmc1 bind to the
ss-DNA
 Accessory factors as Rad54,
Rad54B, and Rdh54 help
recognize and invade the
homologous region
Cont..Cont..

56.  After the formation of
D-loop, DNA
polymerase involved to
elongate the 3’ invading
single strand

57. The recombination repair for a single-strand lesion.
1. DNA replication is stopped at
the lesion but continues on the
opposing undamaged strand
before replisome collapses.
2. Replication fork changes to a
Holliday junction (Chicken
Foot).
3. Single-strand gap at collapsed
replication fork now an
overhang is filled in by Pol I
4. Reverse branch migration
mediated by RuvAB or RecG
yields a reconstituted
replication fork.

58. The recombination repair for a single-strand nick/ Break.
1. Single stranded nick causes
replication fork to collapse.
2. Repair process: RecBCD
and RecA invasion of newly
synthesized and undamaged
3’-ending strand into
homologous dsDNA
3. Branch migration via RuvAB
makes Holliday junction to
exchange 3’-ending strands.
4. RuvC resolves Holliday
junction making the 5’ end
strand nick becomes a 5’ end
of Okazaki fragment.

59. DNA repair of DSBs
via non-homologous recombination repair

61. Scientific Background on the Nobel Prize in
Chemistry 2015
MECHANISTIC STUDIES OF DNA REPAIR
• The Royal Swedish Academy of Sciences has
decided to award Tomas Lindahl, Paul
Modrich and Aziz Sancar the Nobel Prize in
Chemistry 2015 for their “Mechanistic studies
of DNA repair”.

62. The molecular mechanisms of
NER(NUCLEOTIDE EXCISION REPAIR)
• Sancar used the purified UvrA, UvrB, and UvrC proteins to
reconstitute essential steps in the NER pathway.
• The three proteins acted specifically on damaged DNA. With
UV-irradiated DNA as a substrate, the proteins hydrolysed
two phosphodiester-bonds on the damaged DNA strand.
• Later, Sancar could show that the rate of the reaction is
stimulated by UvrD (DNA helicase II) and DNA polymerase I
(Pol I), which catalyses the removal of the incised strand
and synthesis of the new DNA strand, respectively.
• Finally, DNA ligase catalyses the formation of two new
phosphodiester bonds and thus seals the sugar-phosphate
backbone.

64. Discovery of base excision repair
• Based on his observation that uracil is frequently
formed in DNA, Tomas Lindahl came to the conclusion
that there must exist an enzymatic pathway that can
handle this and other types of base lesions.
• He single-handedly identified the E. coli uracil-DNA
glycosylase (UNG) as the first repair protein and two
years later a second glycosylase, specific for 3-
methyladenine DNA.

65. Base excision repair:
• specific base change.
• DNA glycosylase: recognise a
specific type of altered base in
DNA and catalyse its hydrolytic
removal, including: those that
remove deaminated C's,
deaminated A's, different types of
alkylated or oxidized bases, bases
with opened rings, and bases in
which a carbon–carbon double
bond has been accidentally
converted to a carbon–carbon
single bond
• AP endonuclease: cut the sugar
backbone and add in
nucleotides. Depurination can be
therefore directly repaired by AP
endonuclease.

66. • Mammalian cells contain number of different DNA
glycosylases, which act on various forms of base
modifications .
• Once a damaged nucleotide has been identified, the DNA
glycosylase kinks the DNA and the abnormal nucleotide flips
out .
• The altered base interacts with a specific recognition
pocket in the glycosylase and is released by cleavage of
the glycosyl bond.
• The DNA glycosylase itself often remains bound to the abasic
site until being replaced by the next enzyme in the reaction
cycle, the apurinic/apyrimidinic (AP) endonuclease, which
cleaves the DNA backbone at the 5′ side of the abasic
position. The AP endonuclease also associates with DNA
polymerase β (pol-β), to fill the gap.In a final step, DNA ligase
III/XRCC1 heterodimer interacts with pol-β, displaces the
polymerase, and catalyses the formation of a new

67. Mismatch repair
• Modrich developed an assay that allowed analysis of DNA
mismatch repair in cell-free E. coli extracts.
• Modrich could demonstrate that the repair activity was
dependent on ATP, the methylation state of the heteroduplex,
and that mutations affecting mutH, mutL, mutS, and uvrD all
impaired mismatch repair in cell-free E. coli extracts.
• In the paper, Modrich demonstrated the requirement of DNA
polymerase III, exonuclease I, and DNA ligase for
mismatch repair.
• He then combined these factors with purified MutH, MutL,
MutS, UvrD, and single-stranded DNA-binding protein.
Together these factors could process mismatches in vivo in a
strand-specific manner directed by the single, GATC
sequence methylated on only one strand (hemimethylated)
and located distant from the mismatch.

68. Mechanism
of mismatch
correction
repair

69. • MutH binds at hemimethylated GATC sites on the nascent strand.
• MutL acts as a mediator, which interacts with both MutH and MutSboth MutH and MutS. MutL
transduces signals from MutS, which leads to activation of the latent MutH
endonuclease activity causing a nick in the nascent DNA strand near the
hemimethylated GATC-site.
• The machinery now interacts with a helicase (UvrD), which together with
the MutS, MutL, and MutH proteins separates the two DNA strands
towards the location of the mismatch.
• Displacement of the mutant strand continues past, and halts just
downstream of, the mismatch. The nascent strand is then replaced by a
gap-filling reaction, in which DNA polymerase III uses the parental
strand as a template.

70. • In contrast to the situation in E. coli, DNA methylation does
not direct strand specific DNA repair in eukaryotic cells.
• One possibility is that the strand specific nicks formed
during DNA replication can direct strand-specific error
correction.
•
• In support of this notion, mismatch repair is more efficient on
the lagging strand at the replication fork, and a single nick is
sufficient to direct strand specific repair in in vitro.
• Alternatively, the mismatch repair machinery may be directed
by ribonucleotides transiently present in DNA after
replication.