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2. SEMINAR ON EUKARYOTIC
DNA REPLICATION
SUBMITTED BY – DEVENDRA UPRETI
M.SC. II yr

3. DefinitionDefinition
“The process of making an identical copy of a
duplex (double-stranded) DNA, using existing
DNA as a template for the synthesis of new DNA
strands.”
• DNA replication at heterochromatin region and
telomeric region is slow while at euchromatin
region and centromeric dna replication is faster.
• Human 3.0 x 106
• E. coli 4.5 x 103
• Drosophila 1.7 x 105
• Maize 2.0 x 106

4. Chromosomes in Eukaryotic
cells consist of:
DNA
protein
some
chromosom
al RNA

5. G1
preparing for DNA
replication (cell
growth)
S
DNA replication
G2
a short gap before
mitosis
M
mitosis and cell
division
Cell cycle

6. RNA
PROTEIN
DNA
Central
Dogma
Central
Dogma

7. Semiconservativ
e replication
Conservative
replication
Dispersive
replication

8. Experiment of DNA semiconservative replication
"Heavy" DNA(15
N)
grow in 14
N
medium
The first
generation
grow in 14
N
medium
The second
generation

9. DIFFERENCESDIFFERENCES
feature Prokaryote Eukaryote
location Occurs inside the cytoplasm Occurs inside the nucleus
ori one origin of replication per
molecule of DNA
Have many origins of
replication in each
chromosome
Ori length 100-200 or more
nucleotides in length
about 150 nucleotides
Initiation element carried out by protein DnaA
and DnaB
carried out by the Origin
Recognition Complex
DNA polymerase I,II,III α,β,γ,δ,ε

11. BasicBasic rulesrules of replicationof replication
• Semi-conservative
• Starts at the ‘origin’
• Semi-discontinuous
• Synthesis always in the 5'--> 3' direction
• RNA primers required
• In humans occurs at the rate of 3000
nucleotide/minute.
• In bacterial cell it occurs at the rate of 30,000
nucleotide/minute.

12. DNA Replication machinery involved in
Eukaryotes
• DNA Polymerases α,δ,ε,γ.
• Helicase
• Topoisomerase
• Primase
• Ligase
• SSB Proteins
• dNTPs, Mg+2
, & ATP
12

14. DNA Polymerase α
• Involved in initiation
• Synthesizes an RNA primer then adds dNTPs
• A complex of four subunits 50-kD and 60-kD are
primase subunits;180-kD subunit DNA polymerase,
Synthesizes 10-12 nt RNA primers.
• synthesizes RNA primers for the leading strands and
each lagging strand fragments.

15. DNA Polymerase δ
• The principal DNA polymerase ,add okazaki fragments in
lagging strand.
• Has role in proofreading.
• Consists of a 125 kD and a ~50 kD subunit.
• The 50 kd subunit interacts with PCNA (Proliferating Cell
Nuclear Antigen).

16. DNA Polymerase ε:
• Catalyze replication of leading strand ,also
contribute in proofreading.
• DNA Polymerase ε Consist of more than one
subunit, >300 kd
• Polymerases δ and ε both contain th 3‘
5'exonuclease activity required for proof reding.
DNA Polymerase β:
• Monomeric having 36-38 kd Involved in DNA repair
process.
DNA Polymerase γ:
• The DNA-replicating enzyme of mitochondria

17. Proteins Involved in Eukaryotic DNA
Synthesis:
PCNA (Proliferating Cell Nuclear Antigen)
Provide substrate for DNA Polymerase δ, the eukaryotic
counterpart of the Sliding Clamp of E. coli PCNA also encircles
the double helix.
RPA (Replication Protein A)
ssDNA-binding protein that facilitates the unwinding of the
helix to create two replication forks ,the eukaryotic counterpart
of the SSB protein.
RFC (Replication Factor C)
The eukaryotic counterpart of the complex Clamp Loader of E.
coli that is it loads PCNA on DNA .

18. ORC ( Origin recognition complex)
Multisubunit protein, binds to sequences within
replicator Interacts with two other proteins – CDC6 &
CDT1 resulting in loading of MCM complex on DNA .
MCM (Mini chromosome maintenance complex)
Heterohexamer(MCM2-MCM7), ring shaped
replicative helicase, Once the Mcm proteins have
been loaded onto the chromatin, ORC and Cdc6 can
be removed from there.
.

19. DNA ligase
• They are class of enzymes that catalyze the formation of
alpha phosphodiester bond between two DNA chains.
• This enzyme bind OH group at the 3' end of DNA strand
to phosphate group at 5' end of the other.
• linking the two fragments and completing replication of
the region of the lagging strand.

20. STAGES
Initiation
Elongation
Termination

21. – During initiation, proteins bind to the origin of replication
while helicase unwinds the DNA helix and two replication
forks are formed at the origin of replication.
– During elongation, a primer sequence is added with
complementary RNA nucleotides, which are then replaced
by DNA nucleotides.
– During elongation the leading strand is made continuously,
while the lagging strand is made in pieces called Okazaki
fragments.
– During termination, primers are removed and replaced with
new DNA nucleotides and the backbone is sealed by DNA
ligase.

22. initiation
To accomplish this, initiation is partitioned into two
temporally discrete steps:
•Pre replication complex formation during G1 phase
•Activation of pre RC by cdk and ddk enzymes in s phase.

23. Cell Cycle Regulation
• G1 phase of the cell cycle there are low levels of CDK
activity.
• This allows for the formation of new pre-RC
complexes
• During the remaining phases of the cell cycle there
are elevated levels of CDK activity.
• This inhibiting new pre-RC complex formation.
• At the transition of the G1 stage to the S phase of the
cell cycle, S phase–specific cyclin-dependent protein
kinase (CDK) and Dbf4 kinase (DDK) transform the
pre-RC into an active replication fork.

24. Effect of Cdk Activity on pre-RC Formation
and Activation

25. Step in the Formation of the pre-RC
• Recognition of the replicator by the
eukaryotic initiator, ORC (Origin
recognition Complex)
• Once ORC is bound, it recruits two helicase
loading proteins Cdc6 (cell division cycle 6
protein) and cdt1 (chromatin licensing and
DNA replication factor 1 protein).
• ORC and the loading proteins recruite
eukaryotic replication protein i.e. helicase
(the Mcm 2-7 complex)
• Instead the pre-RCs that are formed during
G1 are only activated to initiate replication
after cells pass from the G1 to the S phase of

26. • Phosphorylation of these proteins
results in the assembly to add
additional replication proteins at
the origin and the initiation of
replication at S phase.
• These new proteins include the
three eukaryotic DNA polymerases.
• Then converted to the active
CMG(Cdc45-MCM-GINS) helicase
during S phase.
• OnceDNA Polα/ primase
synthesizes an RNA primer and
briefly extends it. Thus initiation
of replication started.

27. RFC ( replication
factor c) recognizes
primer-template
junctions and loads
PCNA ( proliferating
cell nuclear antigen)
at these sites.

29. Enzymatic activity
• Topoisomerases are responsible for removing supercoils ahead of
the replication fork.
• Helicase unwind the strands at fork.
• Pol α is associated with an RNA primase synthesizing a primer of 10
nucleotide stretch of RNA.
• Pol α for priming synthesis & elongates the newly formed primer with
DNA nucleotides.

30. The Eukaryotic Replication forkThe Eukaryotic Replication fork::
• The progress of the replication fork in eukaryotes is
maintain by helicase activity .
• The separated polynucleotides are prevented from
reattaching by SSBs Proteins.
• The main SSBs Protein in eukaryotes is RPA

32. Leading strand synthesis
• Starts with the primase activity of DNA Pol α to lays down an RNA
primer, then the DNA pol component of Pol α adds a stretch of
DNA.
• RFC assembles PCNA at the end of the primer, PCNA displaces
DNA Pol α.
• DNA polymerase δ or e binds to PCNA at the 3’ ends of the
growing strand to carry out highly processive DNA synthesis
Leading strand synthesis.
• The DNA Polymerase α can extend the initial RNA primer with
about 20 nucleotides of DNA but not capable of lengthy DNA
synthesis.
• After that DNA polymerase δ recognizes this primer and begins
leading strand synthesis in 5′ —> 3′ direction,

33. Priming DNA Synthesis in Bacteria &
Eukaryotes

34. Lagging strand synthesisLagging strand synthesis
• RNA primers synthesized by DNA polymerase α. in the 5′ 3′
direction.
• DNA polymerase d will synthesize short fragments of DNA called
Okazaki fragments which are added to the 3' end of the primer.
• Completion of lagging-strand synthesis requires removal of the RNA
primer from each okazaki fragments.
• To solve this problem there is two models for completion of lagging-
strand synthesis in eukaryotes are describe:
• The RNase H model
• The flap model

35. 36
Rnase H model
ffFlap model

37. • Histone syntesis is continuous threw out the cell
cycle but most amount produced during s phase.
• protein responsible for that are NAP-1 (nucleosome
assembly protein) having a role in to transport
histone from cytoplasm to nucleus.
• other protein is CAF-1(chromatin assembly factor)
that deliver histone to the site of replication .

38. HOW REPLICATION HAS BEEN TAKE
PLACE AT TELOMERIC ENDS....?
TERMINATION

39. • DNA polymaraseDNA polymarase can not replicate the end ofcan not replicate the end of
lagging strand because polymerase requireslagging strand because polymerase requires 3' OHOH
end to bind at daughter strand. so it require someend to bind at daughter strand. so it require some
additional enzyme to perform it.additional enzyme to perform it.
• TELOMERETELOMERE is G - C rich terminal portion ofis G - C rich terminal portion of
chromosome.chromosome.
• Highly repetative and conserved sequenced presentHighly repetative and conserved sequenced present
in eukaryotes.in eukaryotes.
• TELOMERIC sequence in -TELOMERIC sequence in -
 MAMMALS- TTAGGGMAMMALS- TTAGGG
 ARABIDOPSIS- TTTAGGGARABIDOPSIS- TTTAGGG
 PARAMECIUM-TTGGGGPARAMECIUM-TTGGGG

40. Termination of Replication in Eukaryotes
• Removal of the terminal primer at the end of lagging-strand
synthesis leaves a smallgap that cannot be filled in, if left
unfixed, this gap leads to the loss of terminal sequences.This
is known as the end-replication problem. Telomerase (telomere
terminal transferase) is a ribonucleoprotein solve this problem
• Telomerase contains protein and RNA which is complementary
to the DNA sequence found in the telomeric repeat.
Telomerase extend the 3' end of the parental chromosome
beyond the 5‘ end of the daughter strand.
• Telomeric repeat-binding factor (TRF1) and TRF2 bind to
double-stranded telomeric DNA and have a role in telomere
stabilization and telomere-length regulation. The protruding 3′
end can invade the duplex DNA and form a lariat-like structure
called ‘t-loop’, establishing a protective cap .

43. Telomeric activity and agingTelomeric activity and aging
• Telomerase activity is repressed in the somatic cells of
multicellular organism, resulting in a gradual shortening of the
chromosomes with each cell generation, and ultimately cell
death (related to cell aging).
• This telomerase activity find in germ cells and absent in
somatic cell, except cancerous cell.
• In humans due to shortening of telomeric length cause a
inherited diseases of premature aging known as progerious
occur in teen age.

44. Fast & accurate!
• It takes E. coli <1 hour to copy
5 million base pairs in its single chromosome .
• Human cell copies its 6 billion bases & divide into daughter cells
in only few hours with remarkably accuracy.
• DNA polymerase initially makes about 1 in 10,000 base pairing
errors .
• Enzymes proofread and correct these mistakes .
• The new error rate for DNA that has been proofread is 1 in 1
billion base pairing errors .

45. Check points.Check points.
• In order to preserve genetic information during cell
division, DNA replication must be completed with
high fidelity.
• To achieve this task, eukaryotic cells have proteins
in place during certain points in the replication
process that are able to detect any errors during
DNA replication and are able to preserve genomic
integrity.
• These checkpoint proteins inactivate cyclin-
dependent kinases to inhibit cell cycle progression
from G1 to S.

46. Gene Protein Function
ATM
ataxia-telangiectasia
mutated
response to DNA damage
& arrest cell cycle
ATR ataxia- and Rad3-related Arrest cell cycle
PRKDC
DNA-dependent protein
kinase catalytic subunit
(DNA-PKcs)
Arrest cell cycle

47. Telomeric replication
• Telomeres are special nucleoprotein structures composed of double-
stranded (TTAGGG)n DNA repetitive sequence ranging from 3 to 15 kb∼
and a number of telomere associated proteins.
• The ds telomeric DNA terminates at a 3′ single-stranded G-rich
overhang of about 12–500 bases. This protruding 3′ end can invade
the duplex DNA and form a lariat-like structure called ‘t-
loop’.Establishing a protective cap that shields chromosome ends
from being recognized as damaged DNA and prevents nucleolytic
degradation and inappropriate fusions of telomeres.
• The t-loop is stabilized by a complex of ds and ss stranded telomere
binding proteins known as the ‘shelterin’ proteins [TRF1, TRF2]. The
primary role of shelterin is to mark telomeres as the natural
chromosome ends and suppress the DNA damage response pathways
at telomeres

48. Editing & proofreading DNA
• DNA polymerase δ and ε
• proofreads & corrects mistakes
• repairs mismatched bases
• removes abnormal bases
• repairs damage
throughout life
Proofreading refers to any mechanism
for correcting errors in protein or
nucleic acid synthesis that involves
scrutiny of individual units after they
have been added to the chain

50. conclusion
• The process of DNA replication is highly conserved
throughout evolution
• Major replication features in simpler organisms
extend uniformly to eukaryotic organisms
• Thus eukaryotes replicate their DNA in semi
conservative manner
• So, the complete and accurate DNA replication is
integral to the maintenance of the genetic integrity of
all organisms.

51. References
• Greider, C.W. 1996. Telomere length regulation. Annu. Rev.Biochem.
65:337–365.
•
• Rudolph, K.L., S. Chang, H.-W. Lee, M. Blasco, G.J. Gottlieb,C. Greider,
and R.A. DePinho. 1999. Longevity,stressresponse, and cancer in aging
telomerase-deficient mice. Cell.96:701–712.
• M. & McBride, H. Rab,2001, proteins as membraneorganizers. Nature
Rev. Mol. Cell Biol. 2, 107–117.
• D. PETER SNUSTAD, MICHAEL J. SIMMONS,2012 ,Genetics,Jhon wiley
& sons Singapore Pte Ltd, 10,244-249.
• BUCHANAN , GRUISSEM , JONES ,2007, Biochemistry & molecular
bilogy of plants. I.K.International Pvt. Ltd, 06, 260-277.
• Tomaska, L., Makhov, A. M., Griffith, J. D. & Nosek. 2002, J. t-loops in
yeast mitochondria. Mitochondrion 1, 455–459 .
• Anja-Katrin Bielinsky and Susan A. Gerbi . Journal of Cell Science The
Company of Biologists Ltd, 114, 643-651.

52. FOR YOUR PATIENCEFOR YOUR PATIENCE