Download Lec. 2 - DNA replication 1

DNA Replication in Prokaryotes
and Eukaryotes
1. Overall mechanism
2. Roles of Polymerases & other proteins
3. More mechanism: Initiation and
Termination
4. Mitochondrial DNA replication
DNA replication is semi-conservative, i.e., each
daughter duplex molecule contains one new strand
and one old.
Does DNA
replication begin at
the same site in
every replication
cycle?
Electron microscope
image of an E. coli
chromosome being
replicated.
Structure (theta, θ)
suggests replication
started in only one place
on this chromosome.
Fig. 20.9
Does DNA replication begin at the
same site in every replication cycle?
Experiment:
1. Pulse-label a synchronized cell population
during successive rounds of DNA
replication with two different isotopes, one
that changes the density of newly
synthesized DNA (15N), and one that
makes it radioactive (32P).
2. DNA is then isolated, sheared, and
separated by CsCl density gradient ultracentrifugation.
3. Radioactivity (32P) in the DNAs of different
densities is counted.
Prior to 1st replication cycle, 15N (which incorporates into the
bases of DNA) was added for a brief period
st2nd replication cycle, cells were pulsed with 32P
Prior1to
(which gets incorporated into the phosphates of
replicating DNA)
15N - heavy isotope of Nitrogen
32P - radioactive isotope of phosphorus
DNA is isolated,
sheared into
fragments, and
separated by
CsCl-density
gradient
centrifugation.
Blow up of the last
2 rows of DNA in
the previous slide
(i.e., labeled DNA,
and labeled,
sheared DNA).
Same Origin
Random
Origins
Labeled DNA
Labeled,
sheared DNA
Result:
~50% (the most possible) of the
incorporated 32P was in the same
DNA that was shifted by 15N
Conclusion:
Replication of bacterial chromosome
starts at the same place every time
Using Electron Microscopy (EM) to
Demonstrate that DNA Replication is
Bi-Directional
- Pulse-label with radioactive precursor
(3H-thymidine)
- Then do EM and autoradiography.
- Has been done with prokaryotes and
eukaryotes.
Drosophila cells were labeled with a pulse of highly
radioactive precursor, followed by a pulse of lower
radioactive precursor; then replication bubbles were
viewed by EM and autoradiography.
Conclusion: eukaryotic origins also replicate bidirectionally!
Fig. 20.12 in Weaver
Another way to
see that DNA
replication is
Bi-directional -Cleave
replicating
SV40 viral DNA
with a
restriction
enzyme that
cuts it once.
Similar to Fig. 21.2 in Weaver 4
Replicon - DNA replicated from a single origin
Organism
# of replicons
Escherichia coli (bacteria)
1
Average
length of
replicon
4200 kb
Saccharomyces cerevisiae
(yeast)
Drosophila melanogaster
(fruit fly)
Xenopus laevis (frog)
Mus musculus (mouse)
500
40 kb
3,500
40 kb
15,000
25,000
200 kb
150 kb
Homo sapiens
10,000 to
100,000
Š 300 kb
Eukaryotes have many replication origins.
Velocity of
fork
movement
50,000
bp/min
3,600 bp/min
2,600 bp
/min
500 bp/min
2,200 bp
/min
Enzymology of DNA replication:
implications for mechanism
1. DNA-dependent DNA polymerases
– synthesize DNA from dNTPs
– require a template strand and a
primer strand with a 3’-OH end
– all synthesize from 5’ to 3’ (add nt to
3’ end only)
Movie – DNA polymerization
Note: what happens to the P-P?
Comparison of E.coli DNA Polymerases I and III
1 subunit
10 subunits
Proofreading Activity
Insertion of the wrong nucleotide causes the DNA
polymerase to stall, and then the 3’-to-5’ exonuclease
activity removes the mispaired A nt. The polymerase then
continues adding nts to the primer.
Fig. 20.15 in Weaver 4
If DNA polymerases only synthesize 5’ to 3’, how
does the replication fork move directionally?
• Lagging strand synthesized as small
(~100-1000 bp) fragments - “Okazaki
fragments” .
• Okazaki fragments begin as very short 615 nt RNA primers synthesized by primase.
2. Primase - RNA polymerase that
synthesizes the RNA primers (11-12 nt that
start with pppAG) for both lagging and
leading strand synthesis
Lagging strand synthesis (continued)
Pol III extends the RNA primers until the 3’
end of an Okazaki fragment reaches the 5’ end
of a downstream Okazaki fragment.
Then, Pol I degrades the RNA part with its
5’-3’ exonuclease activity, and replaces it
with DNA. Pol I is not highly processive, so
stops before going far.
At this stage, Lagging strand is a series of DNA
fragments (without gaps).
Fragments stitched together covalently by
DNA Ligase.
3. DNA Ligase - joins the 5’ phosphate of
one DNA molecule to the 3’ OH of another,
using energy in the form of NAD
(prokaryotes) or ATP (eukaryotes). It
prefers substrates that are doublestranded, with only one strand needing
ligation, and lacking gaps.
DNA Ligase Substrate Specificity
Ligase will join these two
G--G--A--T--C--C--T--T--G--A--T--C--C
| | | | | | | | | | | | |
C--C--T--A--G G--A--A--C--T--A--G--G
Ligase will NOT join these
two.
G--G--A--T--C--C--T--T--G--A--T--C--C
| | | | |
| | | | | | |
C--C--T--A--G C--A--A--C--T--A--G--G
Ligase will NOT join these
two.
G--G--A--T--C--C--T--T--G--A--T--C--C
| | | |
| | | | | | | |
C--C--T--A--A G--A--A--C--T--A--G--G
Ligase will NOT join these
two.
G--G--A--T--C--C--T--T--G--A--T--C--C
| | | | | |
| | | | | |
C--C--T--A--G G--T--A--C--T--A--G--G
Ligase will NOT join these
two.
C--C--T--A--G
C--T--A--C--T--A--G--G
Mechanism of
Prokaryotic DNA Ligase
Ligase
P
P
HO
5'
3'
NAD
Ligase cleaves NAD and
attaches to AMP.
1
NMN
Ligase-AMP binds and
attaches to 5’ end of
DNA #1 via the AMP.
3'
P
Ligase
1
+
P
3'
AMP
NAD
The 3’OH of DNA #2
reacts with the
phosphodiester shown,
displacing the AMPligase.
NMN
+AMP
2
HO
AMP
+
AMP & ligase separate.
2
1
3'
(Euk. DNA ligase uses
ATP as AMP donor)
P
5'
AMP
Movie - Bidirectional
Replication: Leading and
lagging strand synthesis
Other proteins needed for DNA replication:
4. DNA Helicase (dnaB gene) – hexameric protein,
unwinds DNA strands, uses ATP.
5. SSB – single-strand DNA binding protein,
prevents strands from re-annealing and from
being degraded, stimulates DNA Pol III.
6. Gyrase – a.k.a. Topoisomerase II, keeps DNA
ahead of fork from over winding (i.e.,
relieves torsional strain).
Replisome - DNA and protein machinery at a
replication fork.
DNA Helicase (dnaB gene) Assay
Fig. 20.21 in Weav
Helicase – the movie
Replication Causes DNA to
Supercoil
Rubber Band Model
of Supercoiling DNA
DNA Gyrase relaxes positive
supercoils by breaking and
rejoining both DNA strands.