CHAPTER 12
DNA REPLICATION & RECOMBINATION
Following Watson & Crick’s proposed model
for the structure of DNA, scientists focused
their efforts in determining how the genetic
material replicated itself.
This must be executed precisely if genetic
continuity is to be maintained between cells
following cell division.
Human genome has 3 billion bp within the
23 chromosomes.
An error rate of on one in a million will create
3,000 errors during each replication cycle!
While not error free, the replication mechanism
that has evolved in all organisms is much
more accurate than this.
1
It was clear to Watson & Crick that each
strand of a DNA double helix could serve as a
template for synthesis of its complementary
strand.
The result of such replication would be the
production of two identical double stranded
DNA molecules, each consisting of one “old”
and one “new” strand.
This form of DNA replication is known as
“semiconservative replication”
Two alternative proposed modes of DNA
replication.
[FIGURE 12.1]
2
Meselson--Stahl Experiment (1958)
--Grew E. coli cells for many generations in a
medium where 15NH4Cl was the only nitrogen
source.
--DNA that contains 15N can be distinguished
from 14N-containing DNA through
sedimentation equilibrium centrifugation.
--After many generations, all nitrogen-containing
molecules in E. coli contained 15N.
--Cells were then transferred to a medium
containing only 14NH4Cl.
--All subsequent synthesis of DNA during
replication would now contain the “lighter”
isotope of nitrogen.
--E. coli cells were allowed to replicate during
several generations with cell samples removed
at various time intervals, DNA was isolated
and subjected to sedimentation equilibrium 3
centrifugation. [FIGURE 12.3]
Meselson-Stahl experiment was consistent
with semiconservative reproduction of DNA.
What results would be predicted for conservative
replication?
When the E. coli were grown in 14NH4Cl, there
should be two bands of DNA, one containing
15N and the other containing 14N.
After one round of replication, Meselson and
stahl found a single band containing 15N/14N
thus ruling out this mode of DNA replication.
What results would be predicted for dispersive
replication?
Dispersive replication would result in a DNA
band of itermediate density, as found after the
first round of replication.
4
Meselson & Stahl ruled out dispersive replication
based on the results of 2 experiments.
First, they isolated DNA from the 15N/14N
hybrid molecules and subjected it to heat
denaturation. If dispersive replication was
the mode of DNA replication, then they should
obtain a single hybrid density.
However, they found the single strands exhibited
either an 15N-profile or a 14N-profile thus,
ruling out dispersive replication.
Furthermore, if replication was dispersive, then
all generations after the first, should exhibit a
single band in the density equilibrium
centrifugation. This is not what the data
showed. [FIGURE 12.3]
5
Semiconservative replication is the general mode
by which DNA is duplicated in eukaryotes but
other questions remain.
Where along the chromosome is DNA
replication initiated?
Is there only a single origin of replication?
Is there a specific location on the chromosome
where replication begins or is it random?
Once replication begins, does it continue in a
single direction (unidirectional) or in both
directions away from the origin (bidirectional)?
Results clearly show that there is a single origin
for replication and that replication is
bidirectional!
6
At the actual point along the chromosome
where replication is occurring, the strands of
the helix are unwound, creating a “replication
fork”. With bidirectional replication, two
replication forks are produced.
The length of DNA that is replicated following
one initiation event at a single origin is a unit
called the “replicon”.
[FIGURE 12.6]
DNA synthesis in bacteria requires 3 polymerases
and other enzymes.
Table 12.2
Properties
Initiation of synthesis
5’ - 3’ polymerization
3’ - 5’ exonuclease activity
5’ - 3’ exonuclease activity
Molecules/cell
[FIGURE 12.8]
I
+
+
+
400
II
+
+
?
III
+
+
15
7
DNA replication in bacteria & viruses
In most bacteria and viruses there is a single point
along the circular chromosome for the origin of
replication.
In E. coli, this is a 245 bp region consisting of
Repeating sequences of 9 and 13 bp called oriC.
1. The protein DnaA (encoded by the gene dnaA)
binds to the 9 mers and is responsible for the
initial unwinding of the DNA helix.
2. The binding of DnaA facilitates the binding of
DnaB and DnaC proteins that further open and
destabilize the helix.
DnaA, DnaB, Dna C are called helicases because
they unwind the DNA. Other proteins called
single-stranded binding proteins (SSBPs),
stabilize this conformation
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3. DNA polymerase III requires a primer with
a free 3’ end. A 5 -- 15 bp segment of RNA
complementary to the DNA is synthesized
on the DNA template by an RNA
polymerase called primase.
4. Once the RNA primer is in place, DNA
polymerase III can synthesize the new
strands of DNA in the 5’ to 3’ direction.
Leading strand synthesis
Lagging strand synthesis
Okazaki fragment
DNA polymerease I
DNA ligase
[Figure 12.11, 12.12, 12.13, 12.14].
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DNA replication in Eukaryotes
Similar to prokaryotes but more complex.
1. Multiple Replication Origins along each
chromosome
2. 6 different forms of DNA polymerase
required for DNA replication in eukaryotes:
Polymerase α, β, δ, ε, γ, ζ
3. Difficulties with the ends of linear
chromosomes. [FIGURES 12.18 & 12.19]
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