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DNA Replication
Chapter 9
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How is DNA synthesized?
Parental strand is used as a template for
the newly replicated strand
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3 possible models for DNA
replication
1. Semiconservative
2. Conservative
3. Dispersive
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Experimental evidence for
Semiconservative Model
Meselson and Stahl experiment (1958):
1. Incorporate heavy isotope of nitrogen(15N) into both strands of E. coliDNA,
then allow replication in medium
containing only the light isotope (14N)
2. Separate newly replicated DNA using
density centrifugation
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Predicted results for the 3 models
of DNA replication
(conservative
model
disproved)
(dispersive
model
disproved)
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DNA replication in eukaryotes is
also semiconservative
http://mol-biol4masters.masters.grkraj.org/html/Prokaryotic_DNA_Replication1-Introduction.htm
Herbert Taylors experiment (1958):
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Replicating Chromosome in
E. coli
The E. colichromosome is circular
Preserves the integrity of the circularchromosome
DNA replication initiates at a single
point and proceeds from one or two
replication forks
John Cairns (1963):
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DNA Replication in E. colistarts at an
origin and is bidirectional
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DNA replication in eukaryotes starts at
multiple origins and is bidirectional
Replication bubbles form
at mult ip leor ig inson thelinear eukaryotic
chromosome
replicon: DNA replicated
from a single origin
Drosophi lachromosomes
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DNA Synthesis
Requires DNA Polymerase
1. 3 DNA polymerases : I, II, and III
- polymerase III : replicates most of the DNA
2. Synthesizes DNA in a 5 to 3 direction
3. Synthesizes DNA antiparallel to the parental
strand
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Nucleotide Addition : 5 to 3 Synthesis
Catalyzes formation of
phosphodiester bond
between 5 PO4of
deoxyribonucleotide
and 3 OH on
DNA strand
- substrate is
deoxynucleotide triphosphate
Energy for polymerization comes from cleaving 2
phosphates from deoxyribonucleotide triphosphate
http://en.wikibooks.org/wiki/Medical_Physiology/Cellular_Physiology/DNA_and_Reproduction
5
5
3
5
3
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Nucleotide Addition : 5 to 3 Synthesis
Leading strand is synthesized
continuouslyin direction of
the movement of the
replication forkDNA pol III : high processivity
(synthesizes very long strand)
Lagging strand issynthesizeddiscontinuously(in pieces:
Okazakifragments) in
direction opposite of
movement of replication fork
leading strand
lagging strand
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Leading and Lagging Strand
Synthesis requires primers
Primersare short sequences
of nucleotides (RNA or DNA)
which are base paired to the
template DNA
Primers provide a de novo3OH end for an incoming
deoxyribonucleotide
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Leading and Lagging Strand
Synthesis at the Replication Fork
(~150bp in eukaryotes)
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Lagging Strand Synthesis
Four steps:
1. Primer synthesis
2. Elongation
3. Primer removal with gap filling
4. Ligation
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Primer Synthesis
Primasesynthesizes a primer (10-12 nucleotideslong) complementary to DNA template strand
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Elongation
DNA polymerase III adds deoxyribonucleotides to the3 OH end of the primer
E. coli :
400 nt per second !
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Proofreading activity of DNA polymerase3 to 5 exonuclease activity removes mismatched base pairs
- nuclease: degrades DNA- exonucleasecleaves from end
- endonucleasecleaves phosphodiester
bond between nucleotides
* every polymerase has 35 exonuclease
activity
** only DNA pol I has 53 exonuclease
activity
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Elongation
Fidelity: a measure of polymerase accuracy at incorporating
correct deoxynucleotide(E. coli : 1 mismatch in 109base pairs !)
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Primer Removal and Gap Filling
53 exonucleaseactivity of
DNA polymerase Iremoves primer
53 polymeraseactivity of
DNA polymerase I
fills in the gap with DNA
why is primer made of RNA ?
- primers are not always exact
matches to template
RNA primer can be removedand correct DNA sequence
added by pol III
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Ligation
DNA Ligase seals up nicks left after DNA
polymerase has filled in the gaps
ligase: catalyzes
formation of aphosphodoester
bond from a
5 phosphate
and 3 OH
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DNA synthesis begins at an origin
Step 1: Initiator proteins (dna A) bind origin (ori C)ori C : specific DNA sequence, ~245 bp long
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Binding of initiator proteins at
origin denatures DNA
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HelicaseUnwinds DNA, Primase
Synthesizes a Primer
Helicase + Primase = Primosome
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DNA Polymerase Synthesizes
the Leading Strand
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Primosomes generate more primers
for lagging strand synthesis
C ti d di ti DNA
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Continuous and discontinuous DNA
synthesis occur at replication fork
continuoussynthesisdiscontinuoussynthesis
At li ti f k i
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At replication fork : primosome +
2 DNA pol IIIs + ssb proteins
ssbsbind to single-stranded DNA and keep it from
reannealing during replication
holoenzyme: protein complex with
all associated subunits
DNA pol III : 10 subunits total
- 3 form core enzyme required
for activity
- subunit : clamp that holdspolymerase onto DNA
processivity
replisome:
primosome +
2 DNA pol IIIs
- move as a unit
along DNA
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polymerase cycling :
moving off and on the
lagging strand template
as Okazaki fragments
are completed
for overall movement in
direction of replication
fork :lagging strand must
loop through DNA pol III
to allow 53 synthesis
of Okazaki fragment
away from replication fork
E t t th li ti f k
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Events at the replication fork
during polymerase recycling
2. release of primase3. recruit clamp
1. synthesis of RNA primer
4. recruit DNA pol III,
Okazaki fragment
synthesis 53
5. reattach primase
further along
lagging strand
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Supercoiling
Topoisomerasescan put in or remove supercoils from the DNA
linkage number (L) : number of turns of 1 helix around the other
positive: circular DNA winds around
itself in the same direction asthe twist of helix (right handed)
negative: circular DNA winds around
itself in the opposite direction asthe twist of helix (left handed)
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Topoisomerases
Change the supercoiling of the DNAby increasing or decreasing the
linkage number
Type I Topoisomerases cut one of
the DNA strands, insert unbroken
end in opening
Type II Topoisomerases cut bothDNA strands, insert unbroken
double helix through opening
i.e.,gyrase
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Topoisomerases
Requiredfor DNA replication
As helix opens during DNA replication, positive
supercoils occur in front of the replication forkgyrase removes those supercoils so that
DNA replication can proceed
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Topoisomerase
Termination of
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Termination of
Replication in E. coli
tersites (directly opposite oriC site)
bound by termination protein
(encoded by tusgene : termination
utilization substance)
Intertwined chromosomes have to
be separated by topoisomerase
(type II)
Oth d l t li t
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Other models to replicate
circular chromosomes
1. Rolling Circle
2. D loop
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Rolling Circle Replication in
E. coliPlasmids
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D-LoopReplication in
Mitochondria and Chloroplasts
origins are at different places
on parental template strand
unidirectional leading-strand
synthesis from both strands
(Displacement loop)
R t f DNA S th i d i
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Rates of DNA Synthesis during
replication
E.coli: 25,000 bp/min
eukaryotic : 2000 bp/min
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Eukaryotic DNA Replication
differences from prokaryotes :
9 different DNA polymerases
- : major one in replication
- : makes Okazaki fragment primers
RNA primers removed by RNAse, not DNA pol Multiple origins
- yeast : ARS (autonomously replicating sequences)
Telomeres: special sequences at chromosome ends
similarities to prokaryotes :
specific sequences at origins are bound by proteins
- called Origin Replication Complex (ORC) in eukaryotes
all of the enzymatic processes
How Are the Ends of
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How Are the Ends of
Chromosomes Preserved during
DNA Replication?
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Telomerase
1. Telomerase RNA base pairs with single-stranded 3 end
of DNA
2. Telomerase extends telomere by reverse transcription
(RNA from telomerase is template)
adds GGGGTT (or similar) sequence
3. Telomerase translocates to extended 3 end
RNA + enzyme complex that binds and extends 3 end of linear
chromosomes**ultimate goal : not to increase length, but preserve telomere
several
times
- then primase, polymerase, and ligase make DNA strand
complementary to the new telomere sequence
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Telomerase
1. Telomerase RNA base pairs with DNA
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Telomerase
2. Telomere extension occurs
reverse transcription
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Telomerase
3. Telomerase translocates to extended 3end
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Telomerase
4. Telomerase extends 3 end of telomerereverse transcription
How Telomerase Extends the 3 End
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How Telomerase Extends the 3 End
of a Linear Chromosome
exact size of telomere may
fluctuate from one round
of DNA replication to
the next, but . . .
** genomic information
adjacent to telomere
is preserved**
can be
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Telomere binding proteins
regulate telomeres
TRF1: # of proteins bound to telomeres determines whether
telomerase should extend telomeres
TRF2: prevent end-end fusion of different chromosomes
van Steensel et al., Cell 92::Pages 401413 (1998)
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DNA packaging and replication model
http://www.youtube.com/watch?v=OjPcT1uUZiE&feature=player_embeddedhttp://www.youtube.com/watch?v=OjPcT1uUZiE&feature=player_embeddedTop Related