dna is the genetic material therefore it must 1.replicate faithfully 2.have the coding capacity to...
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DNA is the Genetic Material
Therefore it must1. Replicate faithfully2. Have the coding capacity to
generate proteins and other products for all cellular functioning
• “A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way”.
- Francis Crick
Replication
The Dawn of Molecular Biology
April 25, 1953
Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."
Models for DNA replication1) Semiconservative model:Daughter DNA molecules contain one parental strand and one newly-replicated strand
2) Conservative model:Parent strands transfer information to an intermediate (?), then the intermediate gets copied.The parent helix is conserved, the daughterhelix is completely new
3) Dispersive model:Parent helix is broken into fragments, dispersed, copied then assembled into two new helices.New and old DNA are completely dispersed
(a) Hypothesis 1:
Semi-conservative replication
(b) Hypothesis 2:Conservative replication
Intermediate molecule
(c) Hypothesis 3:Dispersive replication
MODELS OF DNA REPLICATION
Testing Models for DNA replicationMatthew Meselson and Franklin Stahl (1958)
1958: Matthew Meselson & Frank Stahl’s ExperimentSemiconservative model of DNA replication
DNA replication
Replication as a process
• Double-stranded DNA unwinds.
The junction of the unwound molecules is a replication fork.
A new strand is formed by pairing complementary bases with theold strand.
Two molecules are made. Each has one new and one old DNA strand.
DNA Replication
• Since DNA replication is semiconservative, therefore the helix must be unwound.
• John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial circular genome, called an “origin” or “ori” for short.
Origin
5’3’
3’5’
UNIDIRECTIONAL REPLICATION
Origin
5’3’
3’5’
BIDIRECTIONAL REPLICATION
Replication can be Uni- or Bidirectional
John Cairns
Grow cells for several generationsSmall amounts of 3H thymidineare incorporated into new DNA
Grow for brief period
of time
Add a high concentration
of 3H- thymidine
in media with lowconcentration of
3H- thymidine
Bacterial culture
*T
*T
*T
*T
Dense label at the replication forkwhere new DNA is being made
*T*T *T *T
*T*T
*T*T
*T*T*T
*T*T
*T*T*T
*T*T *T *T
*T*T*T*T
*T*T*T
All DNA is lightlylabeled with radioactivity
*T*T *T
Cairns then isolated the chromosomes by lysing the cells very very gently and placed them on an electron micrograph (EM) grid which he exposed to X-ray film for two months.
Evidence points to bidirectional replication
Label at both replication forks
Features of DNA Replication
• DNA replication is semiconservative– Each strand of both replication forks is
being copied.
• DNA replication is bidirectional– Bidirectional replication involves two
replication forks, which move in opposite directions
Arthur Kornberg (1957)
Isolated:Proteins from E. coli
+Template DNA
Added:- dNTPs (nucleotides) all 4 at once- Mg2+ (cofactor)- ATP (energy source)- free 3’OH end (primer)
Synthesized new DNA and isolated a DNA polymerizing enzyme
DNA polymerase I
3’
Kornberg was also able to characterizeHow the DNA polymerase worked!
- dNTPs are ONLY added to the 3’ end of newly replicating DNA
-therefore DNA synthesis occurs only in the5’ to 3’ direction
3’
3’
5’3’5’
5’3’5’
5’3’5’
5’3’5’ 3’
Parental template strandNew progeny strand
THIS LEADS TO A CONCEPTUAL PROBLEM
Consider one replication fork:
5’
3’
5’
3’
Direction ofunwinding
Continuous replication
5’
3’Primer
Primer
5’
3’
Primer
5’
3’Discontinuous replication
Evidence for the Semi-Discontinuous replication model was provided by the Okazakis (1968)
Evidence for Semi-Discontinuous Replication(pulse-chase experiment)
Bacteria arereplicating
Bacterial culture
Add 3H Thymidine
For a SHORT time(i.e. seconds)
Flood with non-radioactive T
Allow replicationTo continue
Harvest the bacteriaat different timesafter the chase
Isolate their DNASeparate the strandsRun on a sizing gradient
smallest
largest
Radioactivity will onlybe in the DNA that was made during the pulse
smallest
largest
Results of pulse-chase experiment
Pulse
5’
3’
5’
3’
Direction ofunwinding
3’
5’
Primer
Primer
5’
3’
Primer
5’
3’
* * *
***
Chase
Continuous synthesis
Discontinuous synthesis
DNA replication is semi-discontinuousDNA Replication is Semi-discontinuous
Continuous Synthesis
Features of DNA Replication
• DNA replication is semiconservative– Each strand of template DNA is being copied.
• DNA replication is bidirectional– Bidirectional replication involves two replication
forks, which move in opposite directions
• DNA replication is semidiscontinuous– The leading strand copies continuously– The lagging strand copies in segments (Okazaki
fragments) which must be joined
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