2/16/16
BIOLOGY 207 - Dr.Locke
Lecture#2 - DNA Structure and Replication
Required readings and problems:
Reading: Open Genetics, Chapter 1
Problems: Chapter 1
Optional:
Griffiths et al. (2008) 9th Ed. Readings: pp 275-291
Problems: 9th Ed. Ch. 7: 1-13,15-25, 28
Campbell (2008) 8th Ed. Readings: Concept 16.1-16.2
Concepts:
How are DNA and chromosomes replicated?
1. The structure of DNA suggests it is replicated semi-conservatively, and this was
supported by the work of Meselson & Stahl
2. DNA is synthesized by enzymes at a replication fork.
3. Cairn's experiments showed chromosomes are semi-conservatively replicated.
4. Eukaryotes have multiple origins and telomerase to solve the "size" and "ends"
problems.
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Lecture#2
Fall'11
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Double helix structure provides a chemical explanation for
the transmission of genes -> (transmission genetics)
Double helix structure explains:
1) - mechanism for the exact replication of genes
- one generation to the next
2) – in chemical terms, the nature of mutations
- cell & organism
3) - the nature of the linear sequence of genes, genes along a chromosome
4)-
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Lecture#2
Fall'11
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Central Dogma – original and modified
Information flow
DNA -----> RNA -----> Protein
Examine one prediction of Watson & Crick Double Helix model
- that of semi-conservative replication
- (not conservative or dispersive)
- each daughter duplex contains one parental and one newly synthesized strand
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Lecture#2
Fall'11
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Meselson & Stahl - 1958
Fig
1. - grew E. coli many generations in medium with
15
N (heavy) not 14N (normal)
- into nitrogen atoms of the bases of DNA
2. - removed from 15N medium
-> 14N and permitted one and two more cell divisions
3. - DNA was extracted from cells and centrifuged at high speed in a CsCI density
gradient to separate DNA with light 14N vs heavy 15N DNA
Meselson & Stahl Observed:
1st generation - single band between 15N + 14N controls
2nd generation - two bands
1 - between 15N + 14N controls
2 - at 14N control
Compare to predictions of various models:
Conservative:
1st generation - 2 bands therefore wrong
Dispersive:
2nd generation - 1 band therefore wrong
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Lecture#2
Fall'11
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Semi Conservative : (correct)
- 1st generation - 1 band
- 2nd generation - 2 bands 1-> 14N
1-> 15N14N mix
Conclusion:
-> evidence that DNA replication is semi-conservative
- refute conservative & dispersive models
-> supports Watson & Crick Double helix model
DNA synthesis
- well described in E. coli - less known about eukaryotes
Rules for chain elongation of DNA by DNA polymerases:
1) Copy already existing chain of DNA - template
2) Growth of the chain is in one direction only 5' -> 3’
3) Need a primer to start DNA synthesis - chain elongation
- DNA pol cannot initiate chain synthesis de novo
4) Duplex DNA replication (replicate both strands) requires a special growing fork
structure
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Fall'11
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At the Replication Fork:
Replication Fork – Fig
Replication Complex (multiple enzymes)
1)-Leading strand
- DNA pol -> continuous synthesis on 3' end - initially started with RNA primer
2)-Lagging strand
- must be synthesized in short discontinuous segments
- each segment consists of RNA primer and replicated DNA (Okazaki fragments)
- repeated cycles permit lagging strand to be synthesized
- polymerase removes RNA primer
- ligase seals the discontinuous fragments into a single strand
RESULT: Both chains replicated and continuous
Both strands are replicated simultaneously:
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Chromosome replication - Prokaryote - Circular DNA
John Cairns' (1963) Experiment
Tested the semi-conservative model using E coli and allowing the cells to replicate and
incorporate (radioactive isotope) 3H-thymidine into DNA)
Hypothesis:
If each newly synthesized daughter molecule is semi-conservatively replicated then
each should contain:
1.- One strand (newly made) that is radioactive
- has 3H-thymidine incorporated - "hot"
2.- Other strand (parental) - contains no 3H-thymidine - "cold"
Cairns Experiment:
- Replicate cells in 3H-thymidine medium for 1 cycle
- Extracted DNA -> spread on a microscope slide
- Put photographic emulsion over top - expose film-> get grain of silver
(Autoradiography)
- Detect location of 3H radiation under light microscope
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Cairns Observed
After 1 replication cycle in 3H-thymidine
- single rings of dots (silver grains).
- See Fig
Cairns interpreted
- these rings as one labeled strand and one unlabeled strand
- evidence that E. coli-bacterial chromosome is circular (confirms previous genetic
analysis)
- no replication forks are seen, or expected to be seen
During the second replication cycle - labeled DNA can be "seen"
1) 1/2 labeled - 1/2 not
2) both labeled
- branch in the ring of dots (silver grains) -> replication forks
- for a circular DNA molecule - see a theta  structure
Density of silver grains is consistent with
- one strand ->1/2 labeled - 1/2 not
- other strand -> both labeled (2 X grains)
- observation of many molecules correspond with the progressive movement of the
replication fork around the ring
Conclusions:
1) Observations are consistent with the semi-conservative model of replication
- applies to whole circular DNA molecules
- Replication forks exist
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2) E. coli parental DNA remains circular throughout the DNA replication cycle
3) Replication - bidirectional? -> not proved here
4) One origin of replication - next section
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Where does the Replication Fork begin?
- Origins of Replication
E. coli origin of replication - See Fig.
- unique - only one in the circular E. coli genome of 4.2 Mb
- called oriC (a gene?)
- has substructure 13 mer + binding sites for protein
Whole chromosome replication starts at fixed Origins of Replication (Ori) and proceeds
bidirectionally (both directions) until replication is finished.
Initiation Process
1) Proteins bind to origin site, unwind the local duplex, and put it in "open"
conformation that is single stranded.
2) Forms a replication fork in both directions
-> bidirectional
3) Replication proceeds around the chromosome and stops at the terminus
-> result is two daughter duplexes
E. coli - chromosome
- bi-directional replication
- replicated in about 40 minutes ~ 4000 kb in size
Therefore each fork replicates 2000 kb in 40 min. or ~ 50 kb/min or ~1000 bases/sec
Prokaryotes have a single replication unit (replicon)
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Lecture#2
Fall'11
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DNA replication in Eukaryotes
Eukaryotes have large genomes with many large linear chromosomes.
Two problems: Size & Ends
Size
How do they replicate their DNA in a short time? Fig.
Multiple origins on the same DNA molecule:
- both ori initiate replication; are bidirectional (2 forks)
- join up - produce long region of replicated DNA
- intermediates sometimes called replication bubbles.
Note:
1) that not every ori need "fire" at the same time - some early - some late
2) ori not all equi-distant - DNA replicated early (near ori ) while some DNA is
replicated late (distant from ori)
3) don't need specific termination sequences.
4) experiments like Cairns shows pattern consistent with bidirectional replication from
many ori sites.
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Lecture#2
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Ends –
Eukaryotic chromosome are linear chromosomes – Telomeres
Replication problem at the end of chromosome!
Fig.
Leading strand - complete replication (no problem)
Lagging strand - no way to get last section replicated
Result -> shortened chromosome each cell generation
Telomerase:
Enzyme called telomerase adds bases to the end.
- consists of tandem arrays of simple DNA sequences.
eg. TTGGGG ->->->->->->
Telomerase adds repeat units
- extends 3' end many repeat units
- primase/polymerase fills in the remaining section
- length is genetically regulated
Fig.
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Lecture#2
Fall'11
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