Lecture PowerPoint to accompany

Molecular Biology
Fourth Edition

Robert F. Weaver

Chapter 20
DNA Replication I:
Basic Mechanism and
Enyzmology
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
 20.1 General Features of DNA
Replication
• Double helical model for DNA includes the
concept that 2 strands are complementary
• Each strand can serve as template for
making its own partner
– Semiconservative model for DNA replication
is correct
– Half-discontinuous (short pieces later stitched
together)
– Requires DNA primers
– Usually bidirectional 20-2
Three Hypotheses of Replication

Three methods of DNA

replication were
considered:
1. Semiconservative
2. Conservative
3. Dispersive

20-3
Semiconservative Replication
• DNA replicates in a semiconservative
manner
• When parental strands separate
– Each strand serves as template
– Makes a new, complementary strand

20-4
 Semidiscontinuous Replication
• DNA replication in E. coli is
semidiscontinuous
• One strand is replicated continuously in
the direction of the movement of the
replicating fork
• The other strand is replicated
discontinuously as 1-2 kb Okazaki
fragments in the opposite direction
• This allows both strands to be replicated in
the 5’3’-direction
20-5
DNA Replication Models

20-6
 Priming DNA Synthesis
• Okazaki fragments in
E. coli are initiated
with RNA primers 10-
12 nt long
• Intact primers are
difficult to detect in
wild-type cells
because of enzymes
that attack RNAs

20-7
 Bidirectional Replication
• The replication structure resembles the
Greek letter, 
• DNA replication begins with the creation of
a “bubble” – a small region where parental
strands have separated and progeny DNA
has been synthesized
• As the bubble expands, replicating DNA
begins to take on the  shape

20-8
 Replicating Forks
• In DNA replication, replicating forks represent
sites of DNA replication
• Is the process:
– Unidirectional – one fork moving away from the other
which remains fixed at the origin of replication
– Bidirectional – two replicating forks moving in
opposite directions away from the origin
• Origin of replication is the fixed starting point for
DNA replication
• Replicon is the DNA under the control of one
origin of replication
20-9
 Unidirectional Replication
• Most eukaryotic and bacterial DNAs
replicate bidirectionally
• ColE1 is an example of a DNA that
replicates unidirectionally

20-10
 Rolling Circle Replication
• Circular DNAs can replicate by a rolling circle
mechanism
– One strand of a dsDNA is nicked and the 3’-end is
extended
– This uses the intact DNA strand as a template
– The 5’-end is displaced
• Phage X174 replication cycles so that when
one round is complete a full-length, single-
stranded circle of DNA is released
• Phage , displaced strand serves as the
template for discontinuous, lagging strand
synthesis
20-11
 Phage Rolling Circle Model
• As the circle rolls right
– Leading strand elongates
continuously
– Lagging strand elongates
discontinuously
• Uses unrolled leading
strand as a template
• RNA primers for Okazaki
fragment
• Progeny dsDNA produced
grows to many genomes
before one genome worth
is clipped off 20-12
 20.2 Enzymology of DNA
Replication
• Over 30 different polypeptides
cooperate in replicating the E. coli DNA
• Examine the activities of some of these
proteins and their homologs in other
organisms
– Start with DNA polymerases – the enzymes
that make DNA

20-13
 E. coli DNA Polymerases
• There are 3 DNA polymerases, the
enzymes that make DNA, found in E. coli:
– pol I
– pol II
– pol III
• E. coli DNA polymerase I was the first
polymerase identified
• It was discovered in 1958 by Arthur
Kornberg
20-14
 DNA Polymerase I
• DNA polymerase I (pol I) is a versatile
enzyme with 3 distinct activities
– DNA polymerase
– 3’5’ exonuclease
– 5’3’ exonuclease
– Mild proteolytic treatment results in 2
polypeptides
• Klenow fragment
• Smaller fragment

20-15
 Klenow Fragment
Contains both: Polymerase and 3’5’
exonuclease activity which serves as proofreading
– If pol I added wrong nt, won’t base pair properly
– Pol I pauses, exonuclease removes mispaired nt
– Allows replication to continue
– Increases fidelity of replication

20-16
 Klenow Fragment Structure
• Wide cleft for binding to DNA between two
a-helices like a hand
– One helix is part of the “fingers”
– Other helix serves as the “thumb” domain
– Between the helices lies a b-sheet, palm
• 3 conserved Asp residues
• Essential for catalysis
• Likely coordinate Mg2+
• Polymerase activity is far separated from
the exonuclease activity 20-17
 5’3’ exonuclease
• This activity allows pol I
to degrade a strand
ahead of advancing
polymerase
• Removes and replaces
a strand in one pass
• Basic functions are:
– Primer removal
– Nick repair

20-18
 Polymerases II and III
• Pol II activity is not required for DNA
replication
• Pol I appears mostly active in repair
• Only pol III is required for DNA replication
– Pol III is the enzyme that replicates bacterial
DNA

20-19
 The Pol III Holoenzyme
• Pol III core is composed of 3 subunits:
– DNA polymerase activity is in the -subunit
– 3’5’exonuclease activity found in -subunit
– Not yet clear what is the role of -subunit
• DNA-dependent ATPase activity is located
in the -complex containing 5 subunits
• Lastly, -subunit plus the other 8 comprise
the holoenzyme

20-20
 Fidelity of Replication
• Faithful replication is essential to life
• DNA replication machinery has a built-in
proofreading system
– This system requires priming
– Only a base-paired nucleotide can serve as a
primer for pol III holoenzyme
– If wrong nucleotide is incorporated
accidentally replication stalls until 3’5’
exonuclease of pol III holoenzyme removes it
• Primers are made of RNA which may help
mark them for degradation 20-21
 Multiple Eukaryotic DNA
Polymerases
Mammalian cells
contain 5 different DNA
polymerases
– Polymerases  and 
appear to participate in
replicating both DNA
strands
– Priming DNA synthesis
is -subunit role
– Elongating both strands
is done by -subunit

20-22
 Strand Separation
• DNA replication assumes that the 2 DNA
strands at the fork somehow unwind
• Does not happen automatically as DNA
polymerase does its job
– 2 parental strands hold tightly to each other
– This takes energy and enzyme action to
separate them
– Helicase that unwinds dsDNA at the
replicating fork is encoded by E. coli dnaB
gene
20-23
 Single-Strand DNA-Binding
Proteins
• Prokaryotic ssDNA-binding proteins bind
much more strongly to ssDNA than to
dsDNA
– Aid helicase action by binding tightly and
cooperatively to newly formed ssDNA
– Keep it from annealing with its partner
• By coating ssDNA, SSBs protect it from
degradation
• SSBs are essential for prokaryotic DNA
replication
20-24
 Topoisomerases
• Strand separation of DNA is referred to as
“unzipping”
– DNA is not really like a zipper with straight, parallel
sides, actually a helix
– When 2 strands of DNA separate, rotate around each
other
• Helicase could handle this task alone if DNA
were linear, short
• Closed circular DNA present special problems
– As DNA unwinds at one site
– More winding must occur at another site
20-25
 Cairns’s Swivel
• A “swivel” in the DNA duplex
called DNA gyrase
• Allows the DNA strands on
either side to rotate to relieve
the strain
• Gyrase belongs to the
enzyme class topoisomerase
• These add transient single-
or double-stranded breaks
into DNA
• Serves to permit change in
shape or topology
20-26