dna is the genetic material therefore it must 1.replicate faithfully 2.have the coding capacity to...
TRANSCRIPT
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