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file:///Users/jlc/Downloads/DNAi_replication_vo2-lg.mov
3x1013 cells (Bianconi et al. Ann Hum Biol, 2013)
1012 replicating cells (Cairns: Genetics (2006)174,1069).
Lose about 1/3 of these per day
30,000 days = life span
If all these cells divide every third day, cells remainingafter 80 years would have undergone ≅ 10,000 divisions
Mutation rate – 10-6/gene/replication
1/100 genes would be mutantIn 1012 cells, 1010 would have mutations, 108 would
have mutations in any pair of genesWhy do so few have cancer?
DNA Replication in vivo
1) Fate of parental DNA
2) Nature of association of template and product
3) Where on the chromosome is DNA replication initiated? Oneor many sites? If many, how are they utilized?
4) Direction of synthesis? Sequential? Bidirectional? Timing?
5) Nature of initation sites
6) Operational definition of leading and lagging strands
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Semi-conservative DNA replication
1) Semiconservative-predicted by W and C
2) Conservative
3) Dispersive
Fate of parental DNA and natureof association with parent
Density transfer-
Substitution of 14N (light) with 15N (heavy) results ina substantial increase in bouyant density- 0.014 g/cm3
Can be measured by density gradient centrifugation-A sedimentation-diffusion equilibrium
DNA is dissolved in CsCl (1.7 gm/cm3). Centrifugeat 100,000 g, generate density gradient- Macromoleculescome to rest at a position corresponding to their density
Meselson-Stahl
Equilibrium CsCl density gradient centrifugation
allows individual DNA molecules to sediment/float to
their own densities in the heavy salt
gradient�Only a duplex of one “all-light” strand and one “all heavy” strand will band tightly at intermediate density
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DNA Replication starts at a fixed point andproceeds sequentially
3) Where on the chromosome is DNA replication initiated? Oneor many sites?
If DNA replication begins at a fixed point, it would look likethis
MARKER FREQUENCY
If marker “a” is near the starting point, then it will be present inthe duplex molecule twice as frequently as marker “b”, near the end.
Other markers will vary from 2 to 1
Needed an assay for “marker frequency”
Transformation
If you have a mutant that cannot grow in medium lacking histidine
Take DNA from wildtype, mix with the mutant
A few cells will take up the DNA, it will recombine withchromosome and cells will grow without histidine-Transformed
Cairns Autoradiographic Experiment
3H thymidine one generation, then put in high specific activity
Spread cells on slides, overlay with film
Study many replicating molecules
DNA replicates as a closed circle
Cairns form or theta form replication
Important point, need a SWIVEL
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? 4) Direction of synthesis? Sequential? Bidirectional?
Spatial and Temporal Organization of DNA Replication in Eukaryotic Cells
• DNA replication in eukaryotes proceeds bidirectionally at multiple and discontinuous subunits along the chromosomal DNA termed ‘replicons.
• There are 30,000-50,000 replicons per mammalian genome with an average size of ~100 kbp.
• Groups of adjacent replicons termed ‘replicon clusters’ replicate synchronously during S-phase. Replicons replicate only once per cell cycle.
• Spatio-temporal organization of replicon cluster synthesis is key to understanding the coordination and choreography of genome replication
Microfluidic assisted replication track analysis:
DNA labeled by growing on nucleoside analogs that can be detected with fluorescently labeled antibodies, spread on PDMS
Sidorova et al. (2009) Nature Methods 4, 839.
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IdU (green, 30 min)+CldU(red, 30 min)
Microarray analysis: 4290 ORFs in E. coli genome
4150 ORFs were made by PCR (polymerase chain reaction)
Array ORFs on a glass slide in ordered array
Carry out DNA replication in synchronized cells (shift dnaC from42 to 30oC. For base line DNA content, take cells at 42oC, label DNA by random priming with Cy3 (green).
R.E. digest DNA from 30oC cells, label by random primed PCR with Cy5 (red) and hybridize to the grid
ORFs that have replicated will be present at about twice theconcentration of unreplicated DNA.
Sample at different times. Measure sequential replication 1/3 rate Stop
Khodursky et al. (2000) PNAS 97, 9419-9124-Analysis of topoisomerase function in bacterial replication fork movement: use of microarrays
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Can also see how far ongoing replication will proceed withouta swivel (Cairns),i.e. in the absence of topoisomerase
Inhibit with novobiocin
10 µg/ml or 500 µg/ml
Synchronize cells so that they initiate together by
arresting at 42oC with a mutationDrop temperature so cells initiate replication in
synchrony Replicate for 30 min, forks half way aroundAdd novobiocin, take sample at various times thereafterMeasure replication of ORFs at various
times after novobiocin addition
ROLLING CIRCLE REPLICATION
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Nature of Initiation Sites
The REPLICON ModelJacob, Brenner and Cuzin, 1963
Replicon - a replicating unit
Replicator - a specific nucleotide sequence that governs whether or not any base sequence attached to it isreplicated - origin of replication
Initiator - encoded by a structural gene, specificallyrecognizes the corresponding origin and acts on it toinduce replication of colinear DNA
E. coli-Initiator, DnaARpelicator,oriC
Isoltation of E. coli origin of replication
Replace a plasmid origin of replication with OriC
plasmid-a DNA that can replicate outside of the chrom-osome but that needs chromosome-encoded gene products-Autonomous replication
Release oriC with a restriction enzyme EcoRI
Ligate to EcoRI treated plasmid encoding ampicillin resistance
Transform E. coli
Select cells that grow in the presence of ampicillin
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oriC plasmids required DnaA protein, a good candidate for theinitiator
Therefore, this was a minichromosome, useful for working outreplication mechanism and regulation
Determine DNA sequence, compare to related, but slightlydiverged bacteria
Manipulate by substitution, deletion and insertion mutations
DNA unwinding elements
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SUMMARY: Replicon Utilization Patterns
Okazaki proposes discontinuoussynthesis to solve the problem ofsequential synthesis of both antiparallel strands
Obligatory intermediates in chaingrowth
ELONGATION
AT A
REPLICATION
FORK is
assymetric
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Pulse labeling of nascent DNA
E. coli thy- [3H]TdR
20oC30oC
Stop by chilling, KCN
[3H]TdR2 sec to 600 sec
Lyse with SDS, denature to release nascent chains
Analyze size
11S, 1000 bases
Simple pulse doesn’t distinguish whether they are precursors or two different populations Ligase minus
Pulse-chase experiment
Are the low mol. wt. fragments precursors of high molecularweight DNA?
E. coli thy- 14C [3H]TdR
20oC30oC
Pulse10 sec
Look at the fate of the label incorporated during the pulseduring subsequent incubation with cold TdR
Distinguishes whether you have two populations or if small converted to large
coldTdR
Chase3 min
Results extended to many systems including animal virus,Yeast, frog- in eukaryotes, only 40-100 bases, however
Both chains are copied discontinously in some systems-wherestrands can be separated and Okazaki fragments canbe hybridized to each
Direction of synthesis, 5’ to 3’, pulse label, digest with nucle-ases
Enzymes involvedPol1- Synthesis but no joining occurs in polA mutantDNA ligase required for joining- in lig mutant, synthesisbut no joining occur
Pol I and ligase are required for maturation
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What determines the size of Okazaki fragments?
What determines where they initiate? Are therespecific sequences?
What enzymes are involved?
TERMINATION OF REPLICATION
Occurs in bacteria at a specialized sequence oppositethe origin on the circular genome
Requires Topo IV for decatenation
Also requires specific sequences called TER anda protein that interacts at TER sites calledTUS, or Ter Utilization Substance
TER sequences form a polar replication block
TUS inhibits the replication helicase
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