Eukaryotic replication

1. Eukaryotic Replication of DNA
Semiconservativereplication,
By- Sanju Sah
St. Xavier’s College, Maitighar
Department of Microbiology
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2. • Native DNA is a double helix made of complementary antiparallel
chains
• DNA can undergo reversible strand separation-denaturation and
renaturation
• DNA is made up of a limited number of monomeric building block
• Polymerization of nucleotides forms nucleic acid-monomers
added one at a time
• Formation of phosphodiester bonds-joins monomers
• Poly nucleotide has a specific start point and fixed terminus- 5’
end to 3’end.
• Molecular template guide the synthesis of macromolecules
• The primary synthetic product is often modified
• DNA replicates before cell divides. Replication is exact and rapid.
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3. 3

4. DNA Denaturation
Denaturation is by
1. Heat: Tm
2. Low ion
concentration
3. Agents destabilizing h-bonds like alkaline solution,
formamid, urea etc
4

5. Template strand
• Melting: Separation of
strands of DNA
two
fully
(replication) or Reversible
partially (transcription)
• Template strand; the separated
strands of DNA work as
template for the synthesis of
DNA or RNA.
• Each strand work as template
to synthesize complementary
strand
• Base pairing is fundamental 5

6. DNA replication begins at an origin
• Replication starts at
specific site: origin of
replication (AT rich region)
where the DNA first
unwind
• Bacterial chromosome with
only one origin
• Origin recognized by
enzyme and DNA separates
in both side forming
replication bubble.
• As replication proceed the
size of bubble increases in
both side- bidirectional
replication
Termination: about 180o of origin
in bacterial circular DNA 6

7. Bidirectional replication of bacterial chromosome
Bidirection
al
The replication may be
• Bidirectional
• Unidirectional
• In virus unidirectional but in
different direction in two origin
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8. One replication of origin of E.coli 8

9. Multiple origins in Eukaryotic chromosome
• E.coli with 4.6 Mbp where as Human with about 6 billion bp in
46 chromosomes.
• E.coli replicate in about 30 min. with same rate it takes 500 hr
to replicate human genome.
• In reality it replicates fast within few hours.
• Replication begins at several origins with several replication
bubbles
• Replicon and replication fork
– About 20 -50 replicons initiate simultaneously
– Euchromatin: replicate early in S-phase, heterochromatin
replicate late in S-phase
– Eukaryote 50bp/s take 30 days to open 105 kb chromosome DNA
with one origin 9

10. Replication starting at different
positions in eukaryotic chromosome
Electron-micrograph
showing multiple replication
bubbles. The arrow shows
the direction of opening
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11. Mechanism
• Similar procedure found in prokaryotic
and eukaryotic cell.
• Replication is governed by complex
formed by proteins in DNA-Replisome
 Multiple replicons in eukaryotes:
Mammalian DNA with 50,000 to
100,000 replicons of 40 to 200 kb. Yeast
DNA has 400 replicons
 Replication requires: Template, DNA
and
polymerase, dNTPs, enzymes
proteins
• Unwinding of helix- only separated
strand work as template
• Binding with polymerase- only at
unwound site.
• Helicase starting from origin separate
two strands by breaking H-Bonds. ATP
works as energy source 11

12. Initiation of replication at E. coli oriC
•Replication bubble with two replication fork running in opposite
directions
•DNA polymerase can bind only at origin of unwinded DNA
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13. • Uncoiling causes Supercoiling
and topoisomerase IV and DNA
gyrase release supercoiling
stress (quinoline antibiotics like
ciprofloxacin
chemotherapy
used in
inactivate DNA
gyrase and inhibit cell division in
cancer cells)
• Single stranded binding protein
(SSB) in prokaryote, replication
protein A (RPA) in eukaryotes
make single strand stable and
prevent base pairing
• In complex eukaryotic
chromosome replication fork
move at about 50 bp/sec-takes
about 30 days to open 108 bp
chromosome.
•50,000 to 100,000
replicons of 40 to 200 kb in
mammelian cell. 13

14. Replication fork
• Single strand DNA is made stabile by
single strand binding protein (SBP) in
prokaryotes. In eukaryotes the re-
annealing is prevented by
replication protein A (ReP A), which
prevent folding of strand and also
prevent base pairing between
same strand.
• The portions of separated strand
works as template for new strand
synthesis
• The replication bubble with two
replication fork opens in two
direction 14

15. DNA polymerase
• In the 1959, Arthur Kornberg and Severo Ochoa shared Nobel
Prize for the discovery of DNA - and RNA Polymerase
respectively in 1950s
• E.coli with 5 DNA polymerases three important are
polymerase I, II, III.
• Pol I for DNA repair during synthesis, Pol II for repair during
damage and Pol III for chain elongation during synthesis.
• Pol III with three units: α-subunit active site with nucleotide
addition, ε-subunit for 3‘ to 5‘ exonuclease activity, the
function of θ-subunit is not known.
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16. DNA polymerase
I II III
1. Gene for the polymerase
subunit
polA polB polC
2. No. of subunits 1 7 > 10
3. Molecular weight (Da) 103,000 88,000 791,500
4. Proofreading (3' -> 5')
exonuclease activity?
yes yes yes
5. 5' -> 3' exonuclease
activity?
yes no no
6. polymerization rate (ntd.
added per second)
16-20 5 - 10 250-1000
7. processivity (nucleotides
added before dissociation)
low
(3-200)
high
(10,000)
very high
(500,000)
DNA polymerase of E.coli 16

17. Eukaryotic Polymerase
• About 15 polymerases
• Pol α: Implicated in repairing DNA, in base excision repair and
gap-filling synthesis, DNA primer synthesis.
• Pol γ: Replicates and repairs mitochondrial DNA and has
proofreading 3'->5' exonuclease activity.
• Pol δ: Highly processive and has proofreading 3'->5'
exonuclease activity. Thought to be the main polymerase
involved in lagging strand synthesis, though there is still
debate about its role
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18. • DNA polymerase α: 4 subunits, Primase activity,
polymerization activity for lagging strand synthesis
• DNA polymerase ε: DNA repair
• DNA polymerase δ: 2 subunits, leading strand synthesis
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Eukaryotic Polymerase

19. Synthesis of DNA
Requirements
• DNA polymerase
• Substrate: template DNA and nucleotides triphosphate
• Protein and enzymes: helicase, SSB, topoisomerase, Mg 2+
Properties of DNA polymerase
• Join two nucleotides during elongation
• Elongates strand in 5’ to 3’ direction, needs free 3’ OH end. Add
nucleotide on free 3’ OH.
• Add nucleotides complementary to template strand
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20. Synthesis of new strand
• The separated strand works as template
• DNA polymerase add nucleotide to only on free 3’OH of pre-
existing polynucleotide DNA or RNA
• Primosome (PriA, DnaG protein) form, a small oligonucleotide
Primer synthesized by primase (Dna G)
• Primer (about 11 to 12 bases) is synthesized at 3’ end of
template
• Polymerase activity is in 5’ to 3’ direction, nucleotide is added
at 3’ OH end (first on primer)
• Phosphodiester bond between 3’ OH of polynucleotide and P of
5’ end of newly added nucleotide
• After several polymerase activity the primosome dissociates
• Replisome: complex controlling replication consists of proteins
bound to open DNA single strand 20

21. Polymerization
reaction
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22. sangerseq.exe
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23. Leading and lagging strand
• DNA synthesis at two template in different directions as the
replication fork opens.
• Leading strand -polymerase reaction in the direction of opening of
fork-one starter primer
• Lagging strand- polymerase activity opposite of the opening of
fork. Multiple primer and Okajaki fragments (1000 to 2000
nucleotides in prokaryote, 100 to 200 ntd in eukaryote)
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24. Replication forming leading and
lagging strands
Reaction joining twoends
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25. • Elongation of leading and lagging strand is coordinated.-
coordination achieved by special protein.
• In absence of such coordination the synthesis of leading and
lagging strands will complete at different times
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26. 26
• The two polymerases of replisome are linked by τ-proteins.
Coordinated polymerase activity leads loop formation by template
of lagging strand.
• The loop increases as the DNA fragment synthesis of lagging strand
elongates.

27. Exonuclease activity of polymerase
• 5’ to 3’ exonuclease activity removes the RNA primer and
polymerase activity fills the gap-both by DNA polymerase I
(classical model)
• Ribonuclease H (RNase H) removes most of the nucleotides of
primer and DNA pol I cleaves remaining few Ntd and add new
nucleotides.
• DNA ligase joins the two ends of two strand
• Replisome contains two polymerase III/δ assembly.
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28. Replication of SV 40 DNA by
Eukaryotic system.
RFC: replicating factor C
PCNC: Ploriferating cell nucleuar antigen
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29. Shortening of lagging strand
during replication –
maintained by Telomerase
• Synthesis of linear DNA at end in
lagging strand shortened at end in each
cycle when the primer is removed
• Telomerase, a modified
transcriptase, elongates the
reverse
lagging
strand from 3’end
• The telomerase has RNA working as
template to add repetitive sequence of
telomere to the end and prevents
shortening 29

30. Replication show high degree of fidelity
• One error in 109 to 1010 nucleotides polymerization in E.coli
• E. coli has 5 × 106 bp genome, so one error in 1000 to 10,000
replication.
• DNA polymerase polymerise one incorrect in 104 to105
• But the Enzymes work on fidelity due to the 3’ to 5’
exonuclease activity
• Proofreading activity: Mismatched occurred during
polymerization is corrected
• Increase accuracy by 102 to 103 fold
• One error in 106 to 108 bases added
• Measured accuracy is still higher (due to mismatch repair
mechanism) 30

31. 3’ to 5’ exonuclease
for proof reading
• The exonuclease
by ε-subunit
activity
of
polymerase. Recognise the
mis-match and cleave it
correct
and add
nucleotide.
• Exonuclease activity only
in new strand, cell has
mechanism to distinguish
new strand and template
strand (methylated A) 31

32. References
• Bhattarai, T: Plant Physiology
• Lodish et al : Molecular cell biology
• Turner et al: Instant notes Molecular biology
• Lehninger: Principles of Biochemistry
• Albrets et al: Molecular biology of cell
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33. • Thank you…
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