broad course objectives for dna replication students will be able to: describe the historic...
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Broad Course Objectives for DNA Replication
Students will be able to:• describe the historic experiment that demonstrated
DNA replication follows a semi-conservative model.• describe the process of DNA replication in
prokaryotes at the biochemical level• explain how proofreading and repair is accomplished
during DNA synthesis
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Outline/study guide—DNA Replication
• At what point in the cell cycle does DNA replication occur?
• When two DNA molecules (or chromosomes) are made from one, where do the parental strands end up, vs. the newly synthesized strands? (i.e. semiconservative replication)
• Why can DNA only be synthesized in the 5’ 3’ direction?
• What are the enzymes and proteins involved in DNA synthesis? What is the function of each and at what point do they act?
• At what point does RNA function in DNA replication?• What determines the lagging strand vs. the leading strand? How does
this change on the “other” side of the replication origin?• How are the Okazaki fragments joined into one continuous DNA strand?
• How does the DNA replication machinery correct errors made during replication?
• Are human chromosomes linear or circular? Bacteria?• Why do linear chromosomes (but not circular chromosomes) have a
problem with telomeres becoming shorter and shorter with each round of replication? How do some cells get around this?
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48 year old woman with Werner Syndrome
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Progeria
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T A
G
C
A
G
A T
T A
T
G
GA
A
C
C
CT
T
G
C G
T A
T A
C
G
A T
T A
C G
T
A T
C G
C
C G
A T
C G
CA
CGG
C
Incomingnucleotides
Original(template)strand
Original(template)strand
Newlysynthesizeddaughter strand
Replicationfork
(a) The mechanism of DNA replication (b) The products of replication
Leadingstrand
Laggingstrand
5′ 3′
3′ 5′
A T
A T
T A
T A
T A
C G
C G
G CG C
G CG C
C G
A T
5′ 3′
5′ 3′
3′ 5′
A T
A T
T A
T A
T A
C G
C G
G CG C
G CG C
C G
A T
3′ 3′
3′ 5′
A T
A T
T A
T A
T A
C G
C G
G CG C
G CG C
C G
A T
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5′3′
A
A T
Brooker Fig 13.1
Identical base sequences
Each strand of the parent DNA
molecule becomes a
template for the new
molecule(s)
The width of the nucleotides reflect larger purines and smaller pyrimidines
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Brooker fig 13.2
Conservative model
Firstreplication
Second replication
OriginalDNA
Semiconservative model Dispersive model
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Different models of DNA Replication
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Brooker fig 13.2
Conservative model
FirstReplication (N14)
Second Replication(N14)
OriginalDNA (N15)
Semiconservative model Dispersive model
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
How do you expect the different models to appear in the centrifuge experiment?
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Brooker fig 13.3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Experimental level Conceptual level
2. Incubate the cells for various cell generations
3. Lyse cells to release DNA
4. Load sample of lysate onto CsCl gradient.
5. Centrifuge until the DNA molecules reach equilibrium densities.
6. View DNA within the gradient using a UV light.
DNACell wall
Cell membrane
Light DNA
Half-heavy DNA
Heavy DNA
(after 2 generations.)
CsClgradient
Lysate
37°C
14Nsolution
Suspension ofbacterial
cells labeledwith 15N
Up to 4 generations
Density centrifugation
Generation0
1
Add 14N
2
1. Grow bacteria in excess of 15N-containing compounds. Switch to 14N at Generation 1.
15N-DNA = purple 14N-DNA = blue
Experiment to distinguish between DNA replication models
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Light
Half-heavy
Heavy
Generations After 14N Addition
4.1 3.0 2.5 1.9 1.5 1.1 1.0 0.7 0.3
*Data from: Meselson, M. and Stahl, F.W. (1958) The Replication of DNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 44: 671−682
Interpreting the Data
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After one generation, DNA is “half-heavy”(consistent with both semi-conservative and dispersive models)
After ~ two generations: DNA is “light” and “half-heavy”(Consistent with which model?)
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Why does DNA (and RNA) only “grow” in the 3’ direction?
New strand Template strand
5’ end 3’ end
Sugar A TBase
C
G
G
C
A
C
T
PP
P
OH
P P
5’ end 3’ end
5’ end 5’ end
A T
C
G
G
C
A
C
T
3’ end
Nucleosidetriphosphate
Pyrophosphate
2 P
OH
Phosphate
Fig from Cambell and Reece, 7th ed(Like Brooker, fig 13-15)
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Brooker, fig 13.10
Origin of replication
Replicationforks
Direction ofreplication fork
1st Okazakifragment
First and second Okazakifragments have beenconnected to each other.
1st Okazaki fragmentof the lagging strand
2nd Okazakifragment
3rdOkazakifragment
Primer
Primer
The leading strand elongates,and a second Okazaki fragmentis made.
The leading strand continues to elongate. A third Okazaki fragment is made, and the firstand second are connected together.
Primers initiate DNA synthesis.Synthesis of the leading strand occurs inthe same direction as movement of thereplication fork. 1st Okazakifragment of lagging strand ismade in opposite direction.
5′
5′
5′
5′
3′
5′
3′
3′
5′
3′
3′
3′
5′
5′
5′
5′
3′
3′
3′
3′
5′
5′3′
3′
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Leadingstrand
DNA strands separate atorigin
Overview of DNA
Replication
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Brooker, fig 13.7
5′3′
5′
5′
3′
3′DNA polymerase III
Origin
Leadingstrand
Lagging strand
Linked OkazakifragmentsDirection of fork movement
DNA polymerase III
RNAprimer
Okazaki fragment
DNAligase
RNA primer
Single-strandbinding protein
DNA helicase
Topoisomerase II
Parental DNA
Primase
Replication fork
• DNA helicase breaks the hydrogen bonds between the DNA strands.
• Topoisomerase alleviates positive supercoiling.
• Single-strand binding proteins keepthe parental strands apart.
• Primase synthesizes an RNAprimer.
• DNA polymerase III synthesizes adaughter strand of DNA.
• DNA polymerase I excises theRNA primers and fills in withDNA (not shown).
• DNA ligase covalently links theOkazaki fragments together.
Functions of key proteins involved with DNA replication
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Brooker, fig 13.5
E. colichromosome
oriC
G GG G GGGA GAGAAAAAA GAA AAT
T
T T ATT TTTA ATTTTTC T TC ATTCT TCCC
1
CC C CCCT CTCTTTTTT CTT TTA A A T AA AAAT TAAAAAG A AG T AAGA AGG
T AG T CCTT AACAAGGAT AGC CAG T T CCT T
T
CGDnaA box
DnaA box
DnaA box
DnaA box
DnaA box
T TGGATCA T CG CTGGA GGA TC A GGAA TTGTTCCT A TCG GTC A A GGA AGCA ACCTAGT A GC GACCT CCA
T CT ACAT GAATCCTGG GAA GCA A A ATT GGAA TCTGAAA A CT ATGTG TA
A
G
C CC C GGTT TACAGCTGG CT
T
T
ATG A A TGA TCGG AGTTACG G AA AAAAC GAAG GG G CCAA ATGTCGACC GT A TAC T T ACT AGCC TCAATGC C TT TTTTG CTT
A GC A TACT GA CGTTCT GTG AGG G T CTA CTCC TGGTTCA T AA CTCTC AAAT CG T ATGA CT AGCAAGA ACCTCC C A GAT GAGG ACCAAGT A TT GAGAG TTT
GA T GTAC CAGTA CA GCA T CAGG CACT A CATG GTCAT GT A CGT A GTCC GT
A GA A TGTA CTT AGGACC CTT CGT T T T AA CCTT AGACTTT T GA T ACAC ATC
AT-rich region
5′ –
–
50
51 100
101 150
201
251 275
250
151 200
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3′
Origin of Replication
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Brooker fig 13.6
AT-rich region DnaA boxes
DnaA proteins bind to DnaA boxes and toeach other. Additional proteins that causethe DNA to bend also bind (not shown).This causes the region to wrap aroundthe DnaA proteins and separates theAT-rich region.
DNA helicase
DNA helicase (DnaB protein) binds to theorigin. DnaC protein (not shown) assiststhis process.
DNA helicase separates the DNA in bothdirections, creating 2 replication forks.
DnaA protein
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5′ 3′
AT- rich
region
ForkFork
3′ 5′
5′3′
5′3′
5′3′
3′5′
3′5′
3′
5′
How the origin sequence initiates
replication
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DNA helicase
DNA helicase separates the DNA in bothdirections, creating 2 replication forks.
ForkFork
5′3′
5′3′
3′5′
3′
5′
Brooker fig 13.6
Travels along the DNA in the 5’ to 3’ direction
Bidirectional replication
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Replication initiation cont.
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Brooker, fig 13.8
3′
3′ exonucleasesite3′
5′ 5′
Fingers
Thumb
DNA polymerasecatalytic site
Templatestrand
Palm
IncomingDNA nucleotides (triphosphates)(dNTPs)
Schematic side view of DNA polymerase III (bacterial)
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Model for how the leading strand and lagging strand coordinate at the replication fork
Brooker Fig 13.12
5′
3′5′
3′
3′
5′ 5′
DNA helicase
Replisome
Primosome
Topoisomerase
Leading strand
DNApolymerase III
Single-strandbinding proteins
Regionwherenext Okazakifragmentwill be made
Primase
RNA primer
New Okazakifragment
Older Okazakifragment
Replicationfork
5′
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Orientation of lagging strand in the replication bubble
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Brooker, ch 13
Chromosome Sister chromatids
Before S phase During S phase End of S phase
Origin
Origin
Origin
Origin
Origin
Centromere(DNA under thekinetochore)
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Eukaryotes have hundreds of origins of replication on their (linear) chromosome
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Replicating DNA of Eukaryotic Chromosomes (Drosophila melanogaster)
Fig from iGenetics, Russell
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Brooker, fig 13.4a
0.25 μm
(b) Autoradiograph of an E. coli chromosome in the act of replication
(a) Bacterial chromosome replication
Replicationforks
Origin ofreplication
Replicationfork
Site wherereplicationends
Cop
yrig
ht
© T
he
McG
raw
-Hil
l Com
pan
ies,
In
c. P
erm
issi
on r
equ
ired
for
rep
rodu
ctio
n o
r di
spla
y.
From Cold Spring Harbor Symposia of Quantitative Biology, 28, p. 43 (1963). Copyright holder is Cold Spring Habour Laboratory Press.
Replicationfork
Bacteria only have one origin on their (circular) chromosome
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Replication rate
• Eukaryotic DNA replication– Typical human chromosome length: 100 million bp– Time to replicate a chromosome: minutes to hours– Hundreds of origins per chromosome– Replicon = ~20,000 to 300,000 bp long– 500-5000 bp / minute at each replication fork
(slower than bacterial replication; that much harder to “unwind” the DNA for replication).
• Bacterial (prokaryotic) replication: – Single circular chromosome (~4.6 million base pairs
[bp])– Single origin of replication single replicon
(“Replication Bubble”)
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Requirements of DNA Replication in a complex organism
• Very low error rate:– One human cell: 6 billion bp of DNA. A
copying error rate of 1 error/million nt 6000 errors with every cell division
• Very fast copy rate– E. coli –1000 nt per minute 3 days to
replicate (real life: 20 minutes per cell cycle; 1000 nt per second)
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Brooker, fig 13.21
DNA polymerase cannot linkthese two nucleotides togetherwithout a primer.
No place for a primer
3′
5′
Linear chromosomes (eukaryotic) cannot easily replicate the ends of chromosomes
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Brooker, fig 13.20
Telomeric repeat sequences
OverhangCG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
TAT G G GA
AT
AT G G GAT AT T T GGG
5′
3′
Chromosome gets shorter at the telomeres with each replication if overhang is left.
In humans and most complex organisms, telomerase is only used in continuously dividing stem cells (e.g. spermatogonia stem cells) most cells get shorter telomeres over time (age). What happened to Dolly, the cloned sheep? (she was generated from a skin cell with shorter telomeres, and she aged early)
Linear chromosomes (eukaryotic) must fill in gap left by RNA primer
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Telomere
Telomerase
Eukaryoticchromosome
Repeat unit
3′
3
5′
T T A G G G T T A
A A T C C C A A TC C C A A U C C C
G G G A G G GT T A T TG G G
T T A G G G T T A
CC C A A U C C C
G G G T T A T T G
GG
T T AG G G A G G G
C C C A A U C C C
TT
C C C A A T A A A AT C C C U A AC UC CC C C
T T A G G G T T A G G G T T A T T GT T AG G G A G G G G G
T T A G G G T T A G G G T T A T T GT T AG G G A G G G G G GT T A G G
A A T C C C A A T
A A T C C C A A T
A A T C C C A A T
RNA
RNA primer
Telomerase synthesizesa 6-nucleotide repeat.
Telomerase moves 6nucleotides to the right andbegins to make another repeat.
The complementarystrand is made by primase,DNA polymerase, and ligase.
3′ 5′5′ 3′
3′
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Brooker, figure 13.22
Step 1 Binding
Step 3 Translocation
The binding-polymerization-
translocation cycle occurs many times
This greatly lengthens one of the strands
Step 2 Polymerization
The end is now lengthened
How telomerase “finishes” the replication of
linear chromosomes
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Go over lecture outline at end of lecture
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Concept Checks
• In the Meselson and Stahl experiment, how was switching the bacterial media from N15 to N14 important for supporting the Semi-conservative model?
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Concept check
• What are the functions of the A-T rich region and DNA boxes in the Origin of Replication?
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Concept Check
• Why is primase needed for DNA replication?
• Is the template strand read in the 5’ to 3’ direction or the 3’ to 5’ direction?
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Concept Check
• Describe the differences between Dna synthesis in the leading strand vs. the lagging strand.
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Which component functions immediately after ligase?
a.Helicase
b.DNA Polymerase 1
c.DNA Polymerase 3
d.primase
e.none of the above
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Which component functions immediately after ligase?
a.Helicase
b.DNA Polymerase 1
c.DNA Polymerase 3
d.primase
e.none of the above