DNA Replication in Prokaryotes and Eukaryotes

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DNA Replication in Prokaryotes and Eukaryotes

• Overall mechanism
• Roles of Polymerases other proteins
• More mechanism Initiation and Termination
• Mitochondrial DNA replication

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DNA replication is semi-conservative, i.e., each
daughter duplex molecule contains one new strand
and one old.
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Figure 20.3
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Electron microscope image of an E. coli
chromosome being replicated. Structure (theta,
?) suggests replication started in only one place
on this chromosome.
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Does DNA replication begin at the same site in
every replication cycle?

• Experiment
• 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).
• DNA is then isolated, sheared, and separated by
  CsCl density gradient ultra-centrifugation.
• Radioactivity (32P) in the DNAs of different
  densities is counted.

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Prior to 1st replication cycle, 15N (which
incorporates into the bases of DNA) was added
for a brief period Prior to 2nd replication
cycle, cells were pulsed with 32P (which gets
incorporated into the phosphates of replicating
DNA) 15N - heavy isotope of Nitrogen 32P -
radioactive isotope of phosphorus
1st
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DNA is isolated, sheared into fragments, and
separated by CsCl-density gradient centrifugation.
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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, sheared DNA
Labeled DNA
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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
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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.

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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
bi-directionally!
Fig. 20.12 in Weaver
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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. 21.6 in Weaver
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Replicon - DNA replicated from a single origin

Eukaryotes have many replication origins.
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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)

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Movie DNA polymerization
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Comparison of E.coli DNA Polymerases I and III
1 subunit 10 subunits
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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.
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If DNA polymerases only synthesize 5 to 3, how
does the replication fork move directionally?
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• Lagging strand synthesized as small (100-1000
  bp) fragments - Okazaki fragments .
• Okazaki fragments begin as very short 6-15 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
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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.
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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
double-stranded, with only one strand needing
ligation, and lacking gaps.
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DNA Ligase Substrate Specificity
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Mechanism of Prokaryotic DNA Ligase- Ligase
binds NAD, cleaves it, leaving AMP attached to
it. Ligase-AMP binds and attaches to 5 end of a
DNA molecule (1) via the AMP. The DNA fragment
with the 3 OH end (2) reacts with the
phosphodiester, displacing the AMP-ligase.
Ligase
NMN
NAD
AMP
N
M
N
3'
P
Ligase

AMP
3'
AMP
(Eukaryotic DNA ligase uses ATP as AMP donor,
instead of NAD).
N
A
D
HO

AMP
P
5'
3'
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Movie - Bidirectional Replication Leading and
lagging strand synthesis
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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.