CHMI 2227E
Biochemistry I
Nucleic acids:
-
replication
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication

The cell cycle defines a
series of events required
for the division of a cell
into two progeny:



S phase: duplication of
DNA
M phase: mitosis
Gap phases (G1 and G2):
preparation for S or M;
DNA
Nucleus
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication

During DNA replication, a
DNA molecule is copied
into 2 progeny molecules
following the basic
Watson-Crick rules:

5’AGCTAGCTGATATCGCGATCG3’
3’TGCATCGACTATAGCGCTAGC5’
Complementarity of the two
strands (i.e. base pairing) 5’AGCTAGCTGATATCGCGATCG3’
3’TGCATCGACTATAGCGCTAGC5’

Antiparallelism (i.e. strands
run in opposite 5’3’
directions);
5’AGCTAGCTGATATCGCGATCG3’
3’TGCATCGACTATAGCGCTAGC5’
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication

Questions to answer:
 1)
Conservation of the parental strands?
 2) Directionality of DNA synthesis?
 3) Nature of the machinery for DNA
synthesis?
 4) Presence of proofreading?
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
1) Conservation of the parental strands

In theory, DNA
replication can occur
through different
mechanisms,
depending on how the
parental strands are
distributed among the
daughter molecules;
Conservative
CHMI 2227 - E.R.
Gauthier, Ph.D.
Semi-conservative
Non-conservative
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Dispersive
DNA replication
1) The Meselson and Stahl experiment
CHMI 2227 - E.R. Gauthier, Ph.D.
PNAS 1958;44;671-682
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DNA replication
2) Directionality of replication

Replication
forks
Studies using electron
microscopy revealed that the
replication of bacterial DNA
molecules occurs bidirectionally:

Both strands are duplicated at
the same time

The site where replication occurs
is called the replication fork;

The parental strand is
denatured at the replication fork;

Replication starts at a specific
point: the origin of replication
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
The replication fork
3’
5’
5’
3’
5’
3’
Direction of the
replication fork
Replication
fork
3’
5’
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
Large DNA molecules can have multiple
origins of replication
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
2) Directionality of replication
BUT: how is replication
preformed with respect to
the polarity of the DNA
strands?

In other words, is DNA
replication occuring:
3’
5’
3’

5’
3’
5’
3’
5’



From the 5’ end towards
the 3’ end, OR
From the 3’ end towards
the 5’ end, OR
Does it matter at all???
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
2) Directionality of replication

In all living organisms,
DNA replication is
always performed in
the 5’3’ direction:
 The
progeny strand is
elongated by the
addition of new
nucleotides at its 3’ end
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
2) Directionality: Okazaki fragments
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
The replication fork
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
3) Nature of the replication machinery

Requirement for DNA replication:
 DNA template
(i.e. the parent DNA molecule);
 All 4 deoxynucleotide triphosphates (dNTPs:
dATP, dGTP, dCTP, dTTP);
 DNA polymerase (an enzyme)
 A DNA or RNA primer to jump start DNA
replication;
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
3) DNA polymerase
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
3) DNA polymerase

The role of DNA polymerase is only to catalyse the formation of the
phosphodiester bond. It has NOTHING to do with base pairing
(well….almost nothing);

Processivity: number of phosphodiester bonds synthesized by the
polymerase before it loses
its -grip
on thePh.D.
DNA molecule.
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CHMI 2227
E.R. Gauthier,
DNA replication
3) DNA polymerase
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
3) DNA polymerase III of E. coli
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
3) DNA polymerase III of E. coli
(Clamp loader)
(DNA polymerase)
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
3) DNA polymerase III of E. coli
http://oregonstate.edu/instruction/bb492/figletters/FigG1.html
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
3) DNA polymerase III of E. coli
Put
DNA
Here!
Sliding clamp
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
3) Primase

DNA polymerase III can only work if
there is a 3’OH available to catalyse
the formation of the phosphodiester
bond;

In other words: before DNA pol can act,
DNA synthesis must already be
initiated…WHAT??

This 3’OH is provided by an RNA
synthetizing enzyme called DNA
primase.

DNA primase synthetizes a short (10
nt) RNA primer complementary to the
DNA template;

DNA pol I replaces the RNA primer by
DNA;
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
Initiation at the origin of replication
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
Leading strand synthesis
Unwinds DNA
http://oregonstate.edu/instruction/bb492/figletters/FigH4.html
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
Lagging strand synthesis

Occurs exactly like leading
strand synthesis, with the
exception that a DNA primase
periodically synthesizes an
RNA primer;
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
Lagging strand
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication - DNA ligase
Lacking
Phosphodiester
bond
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
4) Proofreading
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA replication
4) Proofreading
CHMI 2227 - E.R. Gauthier, Ph.D.
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DNA sequencing
CHMI 2227 - E.R. Gauthier, Ph.D.
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PCR
CHMI 2227 - E.R. Gauthier, Ph.D.
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PCR
CHMI 2227 - E.R. Gauthier, Ph.D.
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CHMI 2227 - E.R. Gauthier, Ph.D.
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