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Eukaryotic Dna Replication 7 and 8
Eukaryotic Dna Replication 7 and 8
Eukaryotic Dna Replication 7 and 8
Chromosomes
are densely
packed in
mitosis
The accuracy of DNA replication is seen
in the quality of the product
Fertilised Egg
Product
Characteristics of ARSs
• Budding yeast replication origins map within such ARS elements on both
chromosomal and plasmid DNA.
B3 B2 B1 ACS
Which proteins bind to and define
Genome, 4 Mb = 4 x 106 bp
Fork rate approx. 800 bp / sec
Replication time approx. 40 minutes = 2,400 secs
Eukaryotes
Interpretation:
Before pulse I:
End of pulse I:
BrdU
c) Stain with anti-BrdU antibodies. BrdU BrdU
BrdU
BrdU BrdU
Duration BrdU BrdU
(hours) ~8 ~2 ~1 BrdU BrdU
template DNA
early-firing origins
late-firing origins
duplicated DNA
The global pattern of origin usage can also change: eg early embryonic
versus somatic cells:
near-synchronous
initiation
Why so many origins?
To allow sections of the genome to replicate faster?
Excess origins are used to lower the probability of a lethal ‘double stall’?
stalled fork
replication completed by
other fork of pair
• Forks generally stop only when they encounter an oppositely moving fork
For example:-
-by regulating the number and spacing of origins that fire eg. during
development
-by regulating the time during S phase at which different origins are activated
Facts II
They are loaded onto DNA in anaphase and are removed from
chromatin during S phase.
Replicon
the unit of DNA in which an individual act of replication occurs.
Origin
site at which replication is initiated.
Terminus
site at which replication stops.
A genome in a prokaryotic cell constitutes a single
replicon; thus the units of replication and segregation
coincide.
A plasmid is an autonomous circular DNA genome that
constitutes a separate replicon; may show single copy
control or under multicopy control. Any DNA molecule
that contains an origin can be replicated autonomously in
the cell.
Each eukaryotic chromosome contains a large number of
replicons; each must be activated no more than once in
each cell cycle.
The DNA of mitochondria and chloroplasts may be
regulated more like plasmids that exist in multiple copies
per bacterium.
Replicons Can Be Linear or Circular
Key Concepts
Key Concepts
Key Concepts
Key Concepts
Key Concepts
http://www.phoenixbiomolecular.com/regenerative_medicine.html
Why do we have them?
• Replication problem
– Lagging strand synthesis
• Unable to replicate the 3’
ends faithfully
• Loose chromosomal DNA
Centromere
Telomere Telomere
Specialized proteins
5’ 3’
Telomeric
t loop
Telomeric
5' proteins:
TRF1
TRF2
TIN2
3' RAP1
NUCLEAR TANKS 1,2
MATRIX POT1
etc
Why are telomeres important?
NHEJ
FUSION
BRIDGE
Mitosis
BREAKAGE
Fusion-bridge-breakage cycles
Genomic instability
5' 3'
3' 5'
5' 3'
5'
5'
3'
http://www.phoenixbiomolecular.com/regenerative_medicine.html
Is Telomerase activity linked to cell immortality?
Telomerase: only in germ, stem and cancer (immortal) cells, but not
in normal cells
Normal
Somatic + Telomerase
10
Cells
(Telomerase
Negative)
kb)
Number of Doublings
Cellular senescence
• Once the telomere shrinks to a certain extent,
the cell stops dividing.
– ~4kb in human cells triggers end to cell division.
• This leads to other changes called cellular
senescence:
– Cell morphology changes.
– Gene expression changes.
Yeast replicative lifespan regulated by telomere length
• Telomerase mutants have a short lifespan: when telomeres shorten to a critical
point, yeast cells stop dividing.
• Overexpression of telomerase:
• Longer telomeres.
• Increased replicative lifespan.
Telomeres in mice
• Lab strains of mice have very long telomeres.
• 30-40kb telomeres.
• Tert knock-out mice:
• Normal for four generations as their telomeres shorten,
• Premature aging phenotypes present in the 5th generation
Telomerase:
Biomedical uses
HYPOTHESIS:
Telomere shortening causes aging and
telomerase will prevent aging
TRUE OR FALSE?
The telomere hypothesis of aging