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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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Telomerase