DNA Replication
Replication always begins at the same place and this place
is the origin
Both eukaryotic and prokaryotic cells must replicate their entire genome once for each and
every cell division. Does replication begin at random places or at specific sites in the genome?
The answer is that replication always begins at specific sites called origins.
Replication bubbles
If DNA from replicating cells is examined
by electron microscopy one can see
replication bubbles. They look something
like this ----->
The replication bubbles are sites at which DNA replication is occurring. The synthesis of new
DNA causes the formation of the bubble.
On the right is shown a replication
fork. As the DNA polymerase
moves, one DNA strand is
converted into two.
Replication Fork
DNA Polymerase
is moving
this way
What does a replicating circle of DNA look like?
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Replicating plasmids were first referred to as theta structures because of their resemblance to
(theta), the eighth letter of the Greek alphabet .
Most cells replicate DNA bidirectionally
In eukaryotic and prokaryotic cells, DNA replication is bidirectional. However, some viruses
employ unidirectional replication. The replication bubbles that I have been drawing could be
produced by one replication fork or by two forks that are moving in opposite directions.
Replication by one fork is called unidirectional replication. Replication by two forks that are
pointed away from each other is called bidirectional replication. Study the following diagrams
until this is clear.
Another description of bidirectional and unidirectional replication
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This diagram represents a
single double stranded DNA
molecule. Let's say that it is a
portion of a bacterial genome.
The black balls represents the
origin of DNA replication.
}
Double
helical
DNA
origin
replication fork
replication fork
Bidirectional Replication
Each strand of the original DNA double
helix is acting as a template for the
synthesis of new DNA (gray). Two
independent sets of enzymes are moving
away from a single origin in opposite
directions (gray arrows) and synthesizing
new DNA in their wake.
Old DNA
Newly synthesized DNA
Unidirectional Replication
Each strand of the original double
helix is still acting as a template for
new DNA synthesis. The difference is
that the replication enzymes are
moving in one direction away from
the origin.
Replicon
A replicon is the region of a chromosome or DNA molecule that is replicated by a single origin.
Bacterial genomes and plasmids are circular and are a single replicon
OK, you know what a genome is, but what is a plasmid? A plasmid is a circular DNA
molecule that replicates inside a cell. It is not considered to be part of the organism's genome
and in fact is dispensable under most circumstances. It is much, much, much smaller than the
genome. There are many different types of plasmids. Most naturally occurring plasmids carry
a handful of genes that confer unique characteristics upon their host cells. For instance, many
plasmids carry the genes for antibiotic resistance, enabling the plasmid-bearing bacteria to
survive and flourish in the presence of an otherwise deadly antibiotic. Other plasmids carry
genes that allow their hosts to consume unusual foodstuffs. Plasmids are of central importance
to the field of molecular biology. More on this later.
The point here is that both bacterial genomes and plasmids are circular DNA molecules
that consist of a single replicon. They have one origin, responsible for replicating the entire
molecule.
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Eukaryotes have linear chromosomes and many replicons
Representation of
a eukaryotic
chromosome with
three replicons.
Each replicon has
its own origin of
DNA replication.
How many origins?
Organism
# of replicons
Escherichia coli (bacteria)
Saccharomyces cerevisiae
(yeast)
Drosophila melanogaster
(fruit fly)
Xenopus laevis (frog)
Mus musculus (mouse)
Homo sapiens
1
500
Average length
of replicon
4200 kb
40 kb
Velocity of
fork movement
50,000 bp/min
3,600 bp/min
3,500
40 kb
2,600 bp /min
15,000
25,000
10,000 to 100,000
200 kb
150 kb
≤ 300 kb
500 bp/min
2,200 bp /min
With regard to this table, appreciate it all but memorize/learn only three things.
1. In general bacterial genomes have a single origin of replication and are therefore a single
replicon.
2. Yeast have many. Flies have many origins.
3. Mammals have lots.
DNA replication rules, rules and more rules
1.
New DNA strands are produced by copying a preexisting DNA strand according to
Watson-Crick base pairing rules. The strand from which the copy is made is called the
template.
The copy is antiparallel and complementary to the template.
2.
All nucleic acids are synthesized in the 5' to 3' direction. This means that the template
strand is read in the 3' to 5' direction.
3.
The enzymes that replicate (copy) DNA are called DNA polymerases.
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Polymerases nomenclature
Polymerases are enzymes that synthesize nucleic acids. The polymerases that replicate DNA
are called DNA-dependent DNA polymerases.
What's in a name?
•
•
•
All polymerases synthesize nucleic acid in the 5'-->3' direction.
No polymerase can synthesize in the opposite direction.
DNA-dependent polymerases require a DNA template which they read in the 3' to
5' direction. They produce an antiparallel and complementary copy of this strand.
All DNA polymerases require a primer sequence which has a free 3' OH. They
extend the strand starting with this 3' OH. This hydroxyl group is on the 3'
position of the sugar moiety. See below.
. . . S P S P S P S P S P S P S
C
A
G
A
T
A
A
T
T
G
G
. . . S P S P S P S
3' OH
. . . S P S P S P S P S P S P S
C
A
G
A
T
A
A
T
T
G
. . .
. . .
G
. . . S P S P S P S
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Prokaryotes have 3 different DNA-dependent DNA polymerases
Why will we be talking so much about bacteria?
We are going to focus on the details of DNA replication in prokaryotes. Why are we paying so
much attention to bacteria? Prokaryotes are simpler and easier to understand and manipulate
than eukaryotic cells. Because of this the puzzle of prokaryotic DNA replication was first solved
in prokaryotes. While eukaryotes are more complex, the big picture underlying events are the
same. The major features of DNA replication are conserved from prokaryotes to eukaryotes.
In prokaryotes there are 3 DNA polymerases. They are DNA polymerase I, II and III. DNA
polymerase I and III participate in DNA replication. DNA polymerase II is involved in SOS
DNA repair.
DNA polymerase I 5’ to 3’ exonuclease
activity Yes 3’ to 5’ exonuclease
activity Yes No Yes DNA Polymerase
III Proofreading Processivity ability Yes low Yes Very high Processivity
In the table above, notice that polymerase I has low processivity while polymerase III has very
high processivity. Low processivity means that the enzyme binds DNA, synthesizes for a short
time and then releases the DNA. High processivity means that polymerase III is tenacious.
Once it begins synthesis it tends to hang on to the DNA and to keep going until the job is done.
T
A
C
A
G
A
T
G
A
Exonucleases remove
nucleotides from the end of
nucleic acid chain.
. . . S P S P S P S P S P S P S
T
What is exonuclease activity?
. . .
. . . S P S P S
. . . S P S P S P S P S P S P S
A
C
A
G
T
T
A
G
. . .
A
T
3'-->5' exonuclease activity
means that the enzyme starts
removing nucleotides from a
free 3' end and proceeds
towards the 5' end.
. . . S P S P S 3' OH
Proofreading
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Both DNA polymerase I and III have proofreading ability. As you will see proofreading and 3'-> 5' exonuclease activity go hand in hand. During synthesis, DNA-dependent DNA polymerase
reads the template strand to decide which nucleotide to add to the growing chain. About once
every 10,000 bases it makes a mistake (1/104 error), an unacceptably high error rate. The
incorrect nucleotide distorts the DNA helix. Sensing this, the polymerase pauses, uses 3'-->5'
exonuclease activity to remove the inappropriate nucleotide, inserts the correct one and then
proceeds on its merry way. Proofreading reduces the error rate of DNA synthesis to about one
mistake per million bases (1/106 error rate). The use of all error correction mechanisms reduces
this to about 10-10 /base.
5ʼ to 3ʼ exonuclease activity
What would a 5ʼ to 3ʼ exonuclease do to this molecule?
. . . S P S P S P S P S P S P S P S P S
A
C
A
G
A
. . . S P S P S
T
T
A
T
G
A
C
T
T
S P S P S P S P S
. . .
A
T
A
. . .
The Replication Fork
The squiggely lines represent newly synthesized DNA. In this picture the bottom strand is the
leading strand and the top strand the lagging strand of DNA synthesis. As the fork moves to
the right it exposes new template that must be replicated. For the leading strand this raises no
difficulty since synthesis is proceeding in the same direction as the fork
(5'-->3'). The DNA
polymerase merely continues to synthesize DNA chasing the fork along.
Problem 1.
A problem arises with the lagging strand of DNA synthesis. In relation to the lagging strand the
fork is moving in the 3'-->5' direction. Because polymerases cannot synthesize in the 3'-->5'
direction it is impossible for the polymerase to synthesize in the same direction as the fork
moves. Therefore, lagging strand synthesis is forced to proceed in the opposite direction to fork
movement (arrow on squiggely line).
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Problem 2.
Now another problem raises its ugly head. Once the fork has moved where does the primer for
DNA synthesis come from?????? The leading strand avoids this problem since it uses the last bit
of synthesized DNA as its primer. The lagging strand can't do this. The solution to this enigma
wrapped in a paradox is in the next section.
At the fork, DNA replication is semi-discontinuous
LAGGING AND LEADING STRANDS ARE VERY IMPORTANT CONCEPTS.
Replication at the fork. Step by Step.
Synthesis of the leading strand begins at the origin. We will talk about this in a later section.
Right now our primary concern is the lagging strand.
In this figure, the rightward movement of
the fork has exposed template that must
be replicated by discontinuous synthesis.
The big black arrow is a primer that has
been made by the enzyme Primase.
Primase is a DNA-dependent RNA
polymerase and its job is to make a primer
for use in lagging strand synthesis. This is
primase's only job.
The primer is about 4 to 15 nucleotides long. Oddly enough, it is an RNA molecule NOT a
DNA molecule. It is complementary to the lagging strand template and is base paired with it.
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DNA polymerase III uses this RNA
primer to begin lagging strand
DNA synthesis.
Usually, a DNA strand of only
about 1000 nucleotides is
synthesized from this primer.
Remember that about 4-15
nucleotides of its 5' end is RNA
while the rest is DNA. This
fragment is called an Okazaki
fragment.
The Okazaki fragment is named
after its discover:
Reiji Okazaki.
In bacteria & phage the Okazaki fragment is about 1000-2000 nucleotides long and takes about 2
seconds to complete.
In eukaryotes it is about 100 -200 nucleotides long.
Reference: Molecular Cell Biology Fourth edition Lodish et al.
After the fork has moved again we
have another exposed region on the
lagging strand template.
And once again Primase makes the
primer and DNA Polymerase III
extends it ------>
Now wait a moment. When extending one Okazaki fragment, DNA Polymerase III will
eventually bump into the 5' end of another Okazaki fragment. What will happen? Well,
Polymerase III will stop synthesizing and leave the lagging strand looking like this:
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The new strand consists of both RNA + DNA. That is, it is just a bunch of Okazaki fragments.
Worse yet, these fragments are NOT even covalently joined to each other. DNA polymerase III
can't join them nor can it remove the RNA. Removal of the RNA is a job for DNA polymerase I.
DNA polymerase I uses the 3' end of an
Okazaki fragment as its primer and
begins synthesis. When it bumps into
the next Okazaki fragment it uses its 5'->3' exonuclease capability to degrade it.
It synthesizes new DNA behind it whilst
degrading the nucleic acid in front of it.
A consequence of this is that it removes
the RNA portions of the next Okazaki
fragment and replaces it with DNA. It
can even remove some of the deoxy
nucleotides from this next fragment
while simultaneously replacing them
with newly synthesized DNA. This is, of
course a waste of energy.
However, because DNA polymerase I has low processivity it soon tires and releases the DNA.
NOTE: There is another way that the RNA moeties can be removed. The enzyme RNase H will
recognize RNA H-bonded to DNA. It will then cleave only the RNA part leaving the DNA part
untouched.
Now the situation has improved. The RNA has been removed from the lagging strand.
However, this strand still consists of many short fragments of DNA. These fragments are
covalently joined with a phosphodiester bond by the enzyme DNA Ligase. This process is
called Ligation.
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Ligation reaction
Ligase catalyzes the formation of a phosphodiester bond between the 5' phosphate of one
molecule and the 3' OH of another molecule. It consumes energy in the form of NAD in
prokaryotes and ATP in eukaryotes and some viruses.
A closer look at the
ligation reaction.
Ligase first binds
NAD and
hydrolyzes it.
Then it is ready to
bind the 5' end of a
DNA molecule.
The DNA is
attached via a
phosphodiester
bond through
AMP.
The DNA fragment
with the free 3' OH
then takes the place
of AMP. A
phosphodiester
bond now exists
between the two
DNA molecules.
Both ligase and
AMP are released.
This is what happens in prokaryotes. In eukaryotes, things are basically the same except that
ATP is used in the place of NAD. That is: ATP--->AMP + PPi.
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Ligase has very stringent substrate requirements.
The two molecules that ligase joins must be base paired with a template strand. Furthermore,
this pairing must be perfect. Finally, the 5' end of one fragment must be adjacent to the 3' end of
the other fragment. That is, there can be no gaps at all.
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
Replisome
All of these bits and pieces, and a few more, actually work together in a loose confederation
which is called the replisome. Lets look at it all together.
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I have mentioned most of these. Let's go over the additions.
Helicase = DnaB
Works at the replication fork. It 'pulls' apart the DNA helix (melts the DNA). DNA polymerase
III nor primase can do this by itself. DNA is a helix and separating the strands will increase the
winding on the rest of the helix. Eventually, torsional stress alone would prevent the fork from
moving forward.
Gyrase (a topoisomerase II)
As stated above the advancing replication fork causes the DNA in front of the fork to become
more tightly wound. Gyrase reduces the resulting torsional stress. It is as if gyrase has inserted
a swivel joint will allows the DNA to spin, reducing the torsion. We will discuss gyrase in more
detail in a later section.
Single-stranded DNA-binding protein or SSB for short
SSB binds to unwound and single stranded template DNA and stabilizes it. It prevents the
double helix from zipping up and from becoming tangled.
Definitions
ATP
lysis
dNTP's
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Abbreviation for adenosine 5' triphosphate. A nucleoside triphosphate composed of
adenine, ribose, and 3 phosphate groups. It is the principal carrier of chemical energy in
cells. Hydrolysis of the terminal two phosphates results in a large release of free energy.
It is NOT the same as dATP.
Rupture of a cell's plasma membrane, leading to the release of cytoplasm and the death of
the cell.
an abbreviation for deoxynucleoside triphosphate, it refers to any or all of the following
dATP, dGTP, dCTP and TTP.
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