Download Chapter 6: DNA Replication and Telomere Maintenance I

Chapter 6: DNA Replication and Telomere Maintenance
I. Introduction
A. Introduction: Reasons For DNA Replication
1. For life to be sustained, cells must divide and give rise to more
cells
2. Each new cell must have a full copy of the genome
3. For some cells, division means reproduction
a. Bacteria
b. Unicellular Fungi
4. Development
5. Wound Repair
B. Introduction: Defects In DNA Replication Have Important
Consequences
1. DNA replication is critical to all life on earth, including all
simple unicellular organisms like bacteria as well as complex
unicellular organisms like humans
a. Important for cell division as each new cell needs a full
copy of the genetic information to function properly
b. Important for reproduction (both mitosis and meiosis)
2. Severe defects in DNA replication will almost always lead to a
loss of survival
3. Other defects that affect DNA synthesis and repair result in
genetic disease
4. One example of a genetic disease linked to defects in DNA
synthesis and repair is Xeroderma Pigmentosum
C. Introduction: Why Replication?
1. It is important before cell division that the whole genome (all
DNA) is replicated
2. Allows for each new daughter cell to have a complete copy of
all DNA sequences
3. In order to do this, replication requires decisions of when to
synthesize the DNA, such that it is done in a complete and
accurate way before the cell starts dividing
II. Mechanisms of DNA Replication
A. Mechanisms of DNA Replication: Introduction
1. When replicating DNA we are taking one double stranded DNA
molecule and making an exact copy of it
2. Based on the double stranded structure there are three
mechanisms by which DNA can be replicated
a. Conservative
b. Semi-conservative
c. Dispersive
3. In conservative replication, there are two products
a. Original double stranded molecule (contains the
original two strands of DNA)
b. The new double stranded molecule of DNA (contains
two newly produced strands)
4. In semiconservative replication, each double stranded DNA
product will consist of 1 newly produced DNA strand and 1
original strand
5. In dispersive replication, some parts of the original helix are
conserved (original DNA) and some parts are newly synthesized
(new DNA)
6. Supplemental Figure: Mechanisms of DNA Replication:
Introduction
B. Mechanisms of DNA Replication: The Meselson-Stahl Experiment
1. In order to determine which of the three mechanisms of DNA
replication were correct, Matthew Meselson, and Franklin Stahl
designed an elegant experiment (1958)
a. Studied DNA replication in E. coli
b. Took advantage of the fact that DNA is nitrogen rich
(nitrogenous bases)
2. Meselson and Stahl grew E. coli in medium containing 15N for
several generations
a. Heavy isotope of nitrogen
b. Over time this isotope gets incorporated into DNA
c. DNA containing 15N is more dense than DNA containing
the normal nitrogen isotope 14N
d. After this treatment, the E. coli had DNA with both
strands containing 15N
3. Next, they shifted the E. coli to media containing 14N
a. Normal nitrogen isotope
b. Did this for only 1 round of replication
B. Mechanisms of DNA Replication: The Meselson-Stahl Experiment
1. Isolated DNA from cells and did density-gradient
centrifugation using a CsCl gradient
a. At the bottom, the concentration of CsCl is high (the
solution is more dense) and at the top, the concentration
of CsCl is low (less dense)
b. Layered their DNA sample on the top of the gradient and
centrifuged the sample
2. During centrifugation, the DNA will be pulled towards the
bottom of the tube by the centrifugal force
3. The DNA will stop moving toward the bottom when it reaches a
concentration of CsCl in the tube of equal density to the DNA
4. DNA Molecules can be observed within the gradient with UV
light at A260
5. If you have conservative replication then you should have a
DNA molecules at the top of the gradient and a double stranded
DNA molecule at the bottom
a. One double stranded DNA molecule contains only 14N
b. One double stranded DNA molecule contains only 15N
5. If you have semi-conservative replication, then you should
have DNA molecules in the center of the gradient
a. One strand of each molecule will contain 14N
b. One strand of each molecule will contain 15N
6. If dispersive replication, then DNA molecules would be located
throughout the gradient
7. Meselson and Stahl saw their DNA run towards the center of
the gradient indicating semi-concervative replication
8. Supplemental Figure: Mechanisms of DNA Replication: The
Meselson-Stahl Experiment
III. DNA Synthesis
A. DNA Synthesis: Introduction
1. In semi-conservative replication, the existing DNA Molecule
will serve as a template
a. Template: Original molecule which serves as a guide to
make a new molecule
b. Each strand will serve as a template
c. The new strand will be complementary to the template
2. DNA synthesis does not happen De Novo (spontaneously), but
requires specifc enzymes called DNA polymerases
a. Multiple DNA polymerases carry out replication
b. DNA polymerase α
c. DNA polymerase δ
d. DNA polymerase ε
3. The DNA polymerases require nucleotides (dNTPs) as
substrates to catalyze synthesis of new DNA
a. Contain deoxyribose
b. Nitrogenous base
c. Three phosphates
B. DNA Synthesis: Addition of Nucleotides to a Growing DNA Strand
1. For all organisms, DNA synthesis occurs in the 5’  3’ direction
a. Nucleotides (dNTP) are added onto the 3’ end of the
growing strand with new phosphodiester bonds being
formed
b. In the condensation reaction, the β and γ phosphates are
lost
2. Any one of four nucleotides can be used for addition onto the
growing DNA chain
a. dATP
b. dTTP
c. dGTP
d. dCTP
3. The choice of nucleotide to add to the growing strand is
determined by complementary base pairing with the template
strand (which is antiparallel)
4. This is why DNA replication is semi-conservative
a. The template strand is from the original double
stranded DNA molecule
b. We are using the template to produce the new strand
C. DNA Synthesis: Prokaryotic vs. Eukaryotic
1. Mechanisms of DNA replication are slightly different in
prokaryotes as compared to eukaryotes
2. The difference in replication mechanisms comes from the fact
that prokaryotic chromosomes are circular, whereas eukaryotic
chromosomes are linear
3. For eukaryotes, the DNA undergoes linear replication
4. For prokaryotes, two methods of DNA replication exist
a. Theta replication
b. Rolling circle replication
5. For all methods, whether prokaryotic or eukaryotic several
basic principles exist
a. DNA replication occurs in the 5’  3’ direction, using a
template that is antiparallel
b. DNA replication begins at sites known as origins of
replication
6. For all methods, whether prokaryotic or eukaryotic several
basic principles exist
a. DNA replication occurs in the 5’  3’ direction, using a
template that is antiparallel
b. DNA replication begins at sites known as origins of
replication
7. DNA replication for eukaryotes, prokaryotes as well as most
DNA viruses is semi-discontinuous
a. One strand is synthesized in the 5’  3’ direction in a
continuous manner
b. One strand is synthesized in the 5’  3’ direction in a
discontinuous manner
IV. Eukaryotic Linear DNA Synthesis
A. Eukaryotic Linear DNA Synthesis: Origins of Replication
1. In Eukaryotes, the chromosomes are linear and quite long
2. The first thing to think about when replicating the
chromosomes is where to start
3. The starting point for DNA replication is at sites that are called
origins of replication
a. At origins of replication, the double stranded DNA helix
is unwound
b. Unwinding creates regions that are no longer double
stranded, but single stranded
c. Each single strand will serve as a template for DNA
replication (DNA replication is semi-conservative)
4. On each human chromosome, it is estimated that there are
between 10,000 and 100,000 origins of replication
5. Human origins of replication lack a consensus sequence, but
are thought to be A-T rich (Have many A-T base pairs)
B. Eukaryotic Linear DNA Synthesis: Unwinding the DNA At Origins of
Replication
1. Now that we have origins of replication, how is it that the DNA
is unwound?
2. Before the DNA is unwound at origins, the histones are first
removed by a yet to be determined process-This loosens the DNA
3. The first step in unwinding of the DNA is the recognition of the
origin of replication by the Origin Recognition Complex (ORC)
4. The ORC will bind to each origin that will be activiated in
replication
5. The ORC is an ATP-regulated DNA binding complex composed
of 6 subunits (ORC 1-6)
6. Once the ORC binds the Origin of Replication, it will recruit two
more proteins
a. Cdc6
b. Cdt1
7. The combined ORC, Cdc6 and Cdt1 complex is considered the
Pre-replication complex
8. The Pre-replication complex will recruit the Mcm2-7 complex
(Mcm stands for mini-chromosome)
9. Once Mcm2-7 complex binds, Cdc6 and Cdt1 dissociate from
the DNA
10. The Mcm2-7 has helicase activity
a. Helicases are enzymes that can act to unwind DNA
b. Once Mcm2-7 acts on the DNA, it is unwound and single
stranded in the region where the origin is
11. Once the DNA is single stranded, the RPA protein will bind the
single stranded DNA to ensure it remains single stranded
12. When the Mcm2-7 complex unwinds the DNA, a replication
bubble forms with 2 replication forks
a. The bubble is the open single-stranded DNA
b. Each fork is the junction where single stranded DNA
meets double stranded DNA
c. The replication fork is where the DNA will be unwound
as DNA replication proceeds
13. Once the DNA is unwound, the Mcm2-7 complex will stay
associated with the DNA
14. Mcm2-7 will move away from the origin as replication
proceeds, creating new areas of single stranded DNA
15. You can think about Mcm2-7 complex moving the replication
forks away from the origin of replication
16. At each origin of replication, there are two forks created that
move in opposite directions which actually create the replication
bubble
C. Eukaryotic Linear DNA Synthesis: DNA Polymerases
1. Once the DNA is single stranded, DNA replication can be
carried out by the enzymes known as DNA polymerases
a. There are three different DNA polymerases that are
involved in eukaryotic replication
b. Each of the DNA polymerases can catalyze formation of
the new strands of DNA only in the 5’3’ direction
2. Three DNA polymerases carry out DNA replication in
Eukaryotes, with each will have a different function
a. DNA polymerase α
b. DNA polymerase δ
c. DNA polymerase ε
3. DNA polymerase δ and ε are the replicative polymerases that
function to add nucleotides onto a growing DNA strand
4. DNA Polymerases are high-fidelity enzymes: they replicate the
DNA without many errors
5. Replicative DNA polymerases are not perfect with mutation
rates ranging from 10-4 to 10-5 per base pair (an error once every
10,000-100,000 base pairs)
6. Replicating DNA polymerases contain a proofreading
exonuclease that can excise 90-99% of misincorporated
nucleotides
7. DNA polymerase δ and ε are the polymerases that function to
add nucleotides onto a growing DNA strand
8. DNA polymerase α is the primase because it functions to lay a
DNA primer
D. Eukaryotic Linear DNA Synthesis: Problems Associated With
Replication Fork Generation
1. Movement of the replication fork machinery causes
supercoiling of the DNA ahead of the fork
2. Supercoiling of the DNA causes torsional stress which could
block replication fork movement
3. Supercoiling ahead of the fork is resolved by topoisomerase I
and II (enzymes that function to unwind DNA)
E. Eukaryotic Linear Replication: Introduction To The Leading and
Lagging Strands
1. If you look at a single replication fork, if DNA polymerase
synthesizes the new strands in the 5’  3’ direction, one strand
will be synthesized away from the replication fork and one
towards the replication
2. The strand that gets synthesized going towards the replication
fork is the leading strand
3. The strand that gets synthesized going away from the
replication fork is called the lagging strand
4. The leading and lagging strands get synthesized in a very
different manner
F. Eukaryotic Linear Replication: Leading Strand Synthesis
1. The leading strand is the easiest strand to synthesize namely
because it occurs continuously
2. The template for the leading strand is the strand that goes
5’3’ away from the replication fork
3. The leading strand is synthesized in the 5’3’ direction
towards the replication fork
4. DNA polymerase ε is thought to be the polymerase involved in
leading strand synthesis, but there may still be a role for DNA
polymerase δ in leading strand synthesis as well
5. To start leading strand synthesis, DNA polymerase ε cannot
bind single stranded DNA and start replication on its own
6. DNA polymerase ε needs a primer with a free 3’OH group to
start synthesis
7. DNA polymerase α (primase) will recognize the single
stranded DNA and lay down an RNA primer
8. This RNA primer will provide the necessary 3’ OH group that
DNA polymerase ε will use to begin synthesis of the leading
strand
9. The DNA polymerase ε will then bind the 3’ OH group and then
catalyze strand synthesis in the 5’3’ direction towards the
replication fork
10. As the replication fork moves away from the origin of
replication, the leading strand will continue to be synthesized,
essentially, into the replication fork
11. As the replication fork keeps moving, DNA can be synthesized
continuously
G. Eukaryotic Linear DNA Synthesis: Lagging Strand Synthesis
1. Synthesis of the lagging strand is more difficult than the
leading strand
2. This is because the lagging strand is synthesized in the 5’3’
direction away from the replication fork
3. This poses a problem because as the replication fork moves,
where do you start the synthesis of the strand?
4. In order to synthesize the lagging strand, this can’t be done
continuously, it must be done in a non-continuous manner
5. The lagging strand is synthesized in fragments that are then
ligated (linked) together
6. The fragments that are put down in lagging strand synthesis
are known as Okazaki fragments
7. The Okazaki fragments are named after Reiji and Tsuneko
Okazaki
8. Reiji is credited for the actual description, however it was
Tsuneko’s work that led to the discovery of the fragments
9. The critical DNA polymerase in lagging strand synthesis is DNA
polymerase δ
10. Like DNA polymerase ε, DNA polymerase δ cannot bind or
start DNA synthesis de novo (on its own)
11. DNA polymerase α will lay down a primer at the site of the
replication fork
12. As the replication fork moves away from the origin of
replication, new single stranded DNA will become available and
another primer will be laid down
13. The addition of each primer will result in the formation of
another fragment of DNA by elongation using DNA polymerase δ
14. Each fragment that is produced is considered an Okazaki
Fragment
15. After each RNA primer is laid down a 3’ OH group is available
a. Allows DNA polymerase δ to bind
b. Catalyze synthesis of DNA
16. For each Okazaki Fragment, DNA polymerase δ synthesizes
DNA in the 5’3’ direction adding nucleotides onto the 3’ end of
the growing strand
17. Addition of new nucleotides results in a phosphodiester bond
between the 3’OH of the previous nucleotide and the 5’ PO4 of the
newly added nucleotide
18. The nucleotide added should have the complementary
nitrogenous base to that on the template strand
19. Each fragment is synthesized by the DNA polymerase ε until
the point where the DNA polymerase δ reaches the next fragment
H. Eukaryotic Linear DNA Synthesis: Piecing Together The Lagging
Strand
1. In order to complete lagging strand synthesis, the Okazaki
Fragments must be pieced together
2. The first step in piecing together the Okazaki fragments is to
remove the RNA primers
3. There are two ways to remove the RNA primer
4. The first mechanism involves RNase H1 nicking the RNA
primer
a. Breaks the hydrogen bonds between base pairs in the
RNA primer and the DNA
b. RNase H1 will leave the last base pair before the RNADNA junction
5. Then, the FEN-1 endonuclease degrades the primer in the
5’3’ direction
a. Has endonuclease activity
b. Has exponuclease activity
6. Then, DNA polymerase δ will fill in the gap between Okazaki
fragments by using the 3’ OH of the upstream fragment to start
gap fill-in
a. Needs that 3’ OH to bind DNA
b. 3’OH is also the place to start adding nucleotides
complementary to template sequence
J. Eukaryotic Linear DNA Synthesis: Piecing Together The Lagging
Strand (Second Model)
1. The second proposed model is simpler than the first
2. In the second model, as DNA polymerase δ is synthesizing an
Okazaki fragment, it will reach the RNA primer of the next
Okazaki fragment
3. When it reaches the next fragment, it will cause displacement
of the RNA downstream RNA primer, and in its place, it will add
nucleotides that are complementary to the template
4. Meanwhile, the FEN-1 endonuclease will then degrade the RNA
primer
5. Note, in this mechanism that the RNase H1 is not required
6. Although it is not entirely clear which of the two mechanisms
is really happening in vivo, data suggests that the real mechanism
is the first
K. Eukaryotic Linear DNA Synthesis: Removing Primer On The Leading
Strand
1. Remember, we also used an RNA primer to start synthesis of
the leading strand
2. This primer must also be removed before and DNA sequence
filled in before leading strand synthesis is complete
3. To remove the RNA primer we must first look at the replication
bubble
4. At the origin of replication, the DNA polymerase ε used in
synthesis of the last Okazaki fragment, will reach the RNA primer
for the leading strand
5. Figure 6.9: Eukaryotic Linear Synthesis: Removing Primer On
The Leading Strand
L. Eukaryotic Linear DNA Synthesis: Making DNA Polymerases More
Efficient By Formation Of The Sliding Clamp
1. Function of the sliding clamp is to increase DNA polymerase
processivity
a. Allows the appropriate replicative DNA polymerase to
add nucleotides over a longer distance
b. Torque caused by the production of the new double
stranded helix would cause the appropriate replicative
DNA polymerase to lose its place at the replication fork
2. In order to solve the problem of the DNA polymerase from
losing its place, a complex called PCNA (proliferating cell nuclear
antigen) acts as a sliding clamp
a. PCNA functions as a trimer (complex of three proteins)
b. Three identical PCNA monomers are joined in a head to
tail arrangement to form a ring shaped structure
3. The DNA double helix is encircled by the PCNA trimer, which
will allow for the DNA polymerase to continually relax and then
regain its hold
4. Without PCNA, torque caused by the production of the new
Double stranded helix would cause the polymerase to lose its
place at the replication fork. PCNA allows the polymerase to
relax and regain its hold
5. A protein complex called RFC places the PCNA onto the DNA
a. Acts as a “clamp loader” and consists of 5 subunits
b. In the presence of ATP, RFC opens the PCNA trimer and
opens the PCNA trimer and passes the DNA into the ring
and then reseals it
c. Structural analysis suggests that RFC may interact with
the minor grooves in DNA
6. Allows For the Polymerization to occur on the order of
thousands of nucleotides
7. Figure 6.7: Formation Of The Sliding Clamp
M. Eukaryotic Linear DNA Synthesis: Termination of DNA Synthesis
1. We’ve synthesized the leading and lagging strands, but the
process must be finished
2. The mechanisms of how replication actually terminates (ends)
are still largely unknown
3. Replication is thought to occur until the next fork is reached
4. Some sequences have been found and shown to be able to halt
replication forks
N. Eukaryotic Linear DNA Synthesis: Replicating The Ends Of The
Chromosomes (Telomeres)
1. To deal with the problem of the ends being shortened after
each round of repliction due to removal of the RNA primer from
the lagging strand, telomeres are created
a. At the completion of lagging strand synthesis there is a
shortened 5’ strand
b. There is a 12-16 nt overhang from the 3’ strand due to
the removal of that 5’ primer
c. Telomeres are known to cap the ends of linear
chromosomes such that they do not remain shortened
2. First identified by Barbara McClintock (1938) working with
maize, and defined by H.M. Muller in Drosophila
3. Telomeres are comprised of tandem repeats of a guanine (G)
rich sequence
4. Sequence is evolutionarily conserved from yeast to ciliates to
plants to mammals
5. Human telomeres contain thousands of repeats of the
sequence TTAGGG, whereas tetrahymena has telomere sequence
TTGGGG
O. Eukaryotic Linear DNA Synthesis: Creation of Telomeres
1. Telomeres maintain chromosomal length and seal the ends of
chromosomes
2. Confer stability by keeping the chromosomes from ligating
together
3. Loss of telomeres leads to end-to-end chromosome fusions,
facilitates genetic recombination and triggers cell death
P. Eukaryotic Linear DNA Synthesis: Solutions To The End Replication
Problem – Discovery Of The Telomerase Enzyme
1. McClintock discovered telomeres at chromosomal ends,
however it was still unknown how this was the solution to the
end replication problem for the lagging strand
2. Greider and Blackburn (1989) discovered how telomeres were
added by working with the small single-celled Eukaryote
Tetrahymena thermophila
a. Tetrahymena has many chromosomes
b. Tetrahymena will have many telomeres (40,000) (Note
that there can be more than two per chromosome
c. Expected that the machinery that added telomeres
would be abundant in these organisms
d. The same year telomerase activity was found in human
cells
3. Greider and Blackburn discovered the enzyme terminal
transferase (Telomerase) which catalyzed de novo addition of
telomeric repeats to the end of chromosomes
a. Telomerase enzyme is a ribonucleoprotein (RNP)
complex (The enzyme has an RNA and a protein
component)
b. The first component purified was the telomerase RNA
component called TERC (template for telomerase RNA
compoent)
c. Each subunit of telomerase has an important function
The RNA component functions as a template for telomere
addition
d. The protein component functions as a reverse
transcriptase
4. Figure 6.20: Eukaryotic Linear DNA Synthesis: Solutions To
The End Replication Problem – Discovery Of The Telomerase
Enzyme
Q. Eukaryotic Linear DNA Synthesis: Mechanism Of Telomere Addition
1. Surprisingly, telomerase does not extend the short 5’ (lagging
strand)
2. Telomerase instead interacts with the 3’ end of the lagging
strand template
a. Causes elongation of the 3’ template of the lagging
strand
b. Adds one telomere repeat at at time
c. Repositioning allows for further additions of repeats
(one at a time)
d. Addition occurs in the 5’  3’ direction by the
telomerase enzyme
3. Once the telomerase has added telomere to the 3’ end of the
lagging strand template, synthesis of the lagging strand end can
occur
a. DNA polymerase α can then lay an RNA primer
b. DNA polymerase ε performs the elongation step
R. Eukaryotic Linear DNA Synthesis: Regulation of Telomere Length
1. Although telomeres are important in maintaining appropriate
chromosome length, it is also important that they do not become
too long (length is regulated)
2. Three proteins play a role in maintaining the appropriate
telomere length
a. POT1
b. TRF1
c. TRF2
3. POT1 (protection of telomeres) binds the 3’ end single
stranded DNA tail while TRF1 and 2 (TTAGGG repeat binding
factors) prevents telomerase access by forming a folded
chromatin structure
4. It is unknown how this system functions to sense telomere
length
5. It is clear that the amount of POT 1 present acts as a sensor
(when enough POT1 is present) this leads to a shut down
telomere addition
S. Eukaryotic Linear DNA Synthesis: Telomerase, Aging And Cancer
1. In most uni-cellular organisms, telomerase has a housekeeping
function – core components are always expressed
2. Human cells that do not divide rapidly do not produce high
quantities of telomerase and cannot maintain telomere length
during cycles of replication – Each round of cell division for these
cells, the telomeres get shorter
3. Rapidly dividing cells maintain high level of telomerase, and
telomere length does not shorten
4. Telomere shortening is a proposed “molecular clock” that
serves to count the number of cell divisions
5. Once the telomeres reach a certain length (shortened),
proliferative arrest occurs (senescence): usually at about 20
divisions
T. Eukaryotic Linear DNA Synthesis: Telomerase, and aging- Two
Experiments Show The Connection Between Telomerase And Aging
1. Experiment 1: Knockout of the telomerase RNA
a. Wild-type cells can divide well up to 450 divisions (note:
mice telomeres are longer, and it takes 300 divisions, for
mouse cells to reach senescence)
b. Telomerase deficient mouse cells may stop dividing as
only about 6 divisions, and premature aging was observed
2. Experiment 2: The reverse transcriptase (hTERT) component
of the telomerase is overexpressed in human somatic cells (Note:
the reverse transcriptase component is limiting)
3. Overexpression of telomerase results in immortalization
4. Figure 6.22 Eukaryotic Linear DNA Synthesis: Telomerase, and
Aging- Two Experiments Show The Connection Between
Telomerase And Aging
V. Prokaryotic DNA Replication
A. Prokaryotic DNA Replication: Introduction
1.Prokaryotes commonly have two types of DNA molecules
a. Basically all bacteria have a circular chromosome
containing most if not all of their genes
b. Some may contain plasmids-the plasmids are small
circular DNA molecules that can contain genes conferring
antibiotic resistance
2. There are two different mechanisms by which bacteria can
replicate their DNA
a. Rolling Circular Replication (Plasmids)
b. Bi-Directional Replication (Theta ReplicationChromosomes)
c. Uni-Directional Replication
B. Prokaryotic DNA Replication: Rolling Circle Replication
1.. Rolling circle replication is used to replicate some plasmids
2. Plasmids are small circular DNA molecules that are double
stranded
3. For rolling circle replication, the outer strand is considered the
(+) strand and the inner strand is the (-) strand
4. To start rolling circle replication, the (+) strand is nicked (cut)
5. In rolling circle replication, the (+) strand serves as a template
for the (-) strand and the (-) strand serves as a template to make
the (+) strand
6. The nick in the (+) strand will create a free 3’ OH and allow for
synthesis of a new (+) strand
a. Synthesis of a new (+) strand will start at the free 3’OH
using the (-) strand as a template
b. Synthesis of a new (+) strand is continuous lending the
name rolling circle because we go around the circle
7. Synthesis of a new (-) strand uses the (+) strand as a template
8. Synthesis of a new (-) strand occurs in a discontinuous
replication
C. Prokaryotic DNA Replication: Bi-Directional Replication In Bacteria
(Theta Replication) Introduction
1. Bi-direction DNA replication is also known as theta replication
2. It is called theta replication because the intermediate created
during DNA synthesis look like the Greek letter theta
3. In Bi-Directional replication, two replication forks are created
at an origin of replication, just as in eukaryotic replication with
BOTH forks moving as the DNA is being replicated (similar to
eukaryotes)
4. Replication ends as the forks meet each other at the other end
of the circular chromosome (Prokaryotes only)
D. Prokaryotic DNA Replication: Evidence For Two Replication Forks In
Bacteria
1. John Cairns is an English Molecular Biologist, who first
uncovered evidence for two replication forks in Bacterial
chromosome replication
2. John Cairns worked with replicated E. coli chromosomal DNA
3. E. coli chromosomal DNA is circular
4. He labeled replicating E. coli DNA with a radioactive nucleotide
for one full round and part of a second round of replication
5. After the first round, the DNA should have two strands
a. One strand is radiolabeled (new)
b. One strand is not radiolabeled (old-template)
E. Prokaryotic DNA Replication: Evidence For Bi-Directional Replication
In Bacteria
1. Although there are two replication forks created, there are two
possible ways that the circular Bacterial chromosome could be
replicated
a. Uni-directional (DNA is replicated at only one of the
forks)
b. Bi-Directional (DNA is replicated at both forks)
2. Elizabeth Gyurasits and R.B. Wake showed that DNA
replication in B. subtilis is bi-directional
3. Grew B. subtilis in the presence of 3H-Thymidine (weak
radioactivity) for a short time
4. Then switch the B. subtilis to media with a more strongly
radiolabeled nucleotide (32P)
5. Exposed the replicating DNA to autoradiography
6. Both forks picked up the strongly radiolabeled nucleotide, and
must have been replicating at the point where the shift occured
F. Prokaryotic DNA Replication: Evidence For Two Replication Forks In
Bacteria
1. For the next round of replication, when the DNA is unwound,
one template strand will be radiolabeled and the other strand
will not
2. Therefore, when replicating in the second round one newly
forming double stranded DNA molecule will have one strand
labeled, and the other newly forming DNA molecule will have
both strands labeled.
3. The structure formed during the second round of replication
looks like a theta (θ)
4. The results from the newly forming molecule with two
radiolabeled strands showed that there were two replication
forks
G. Prokaryotic DNA Replication: Starting Bi-Directional Replication
1. Just like in eukaryotes, replication must occur at an origin of
replication
2. Unlike in Eukaryotes, there is only going to be one origin of
replication
a. The bacterial chromosome is circular
b. The bacterial chromosome is significantly smaller than
a eukaryotic chromosome
3. In the E. coli chromosome, the origin of replication has a
specfic name
a. The origin of replication is called OriC
b. OriC has four 9-mers of the sequence TTATCCACA and
three 13-mer repeats
4. The first step in Prokaryotic chromosomal replication is to
unwind the DNA
5. To unwind the DNA, initiator proteins will bind the OriC and
unwind a small section of the DNA
6. Then a protein called DNA helicase will further break
hydrogen bonds existing between base pairs the two strands to
unwind the DNA (note: DNA helicase will move in the 5’  3’
direction along the lagging strand template)
7. Once the DNA is unwound, two replication forks are created
going in opposite directions
8. When the DNA is unwound, then there are single stranded
templates to use for replication
H. Prokaryotic DNA Replication: Unwinding the DNA and Building A
Primosome Complex At The Origin Of Replication
1. The DNA is unwound at the OriC and requires several proteins
a. DnaA
b. DnaB (helicase)
c. DnaC
2. The nine-mers of OriC function as a binding site for DnaA
3. Once DnaA binds, it will then help facilitate the binding of
DnaB
a. DnaA helps DnaB bind the origin by stimulating the
melting of the three 13-mer repeats just to the left of OriC
b. DnaB will also need the aid of DnaC to bind the OriC
c. DnaB is a helicase, and catalyzes the formation of two
replication forks at an origin of replication
d. DnaB requires ATP to catalyze formation of replication
forks
4. This protein complex, plus the RNA polymerase and HU
protein form the primosome complex
J. Prokaryotic DNA Replication: Bi-Directional Replication and the
Replicative DNA Polymerases
1. Once the DNA is unwound, replication occurs in much the same
way as it does in Eukaryotes (with both leading and lagging
strands)
a. Proteins mediate the process of replication although
they are different than in eukaryotes
b. Nucleotides to serve as raw materials to make the DNA
c. Single stranded DNA serves as a template (The
replication is semi-conservative)
2. Just like in eukaryotic replication DNA polymerases mediate
the synthesis of the new DNA
a. DNA polymerase I (pol I) fills in the gaps between
Okazaki Fragments
b. DNA polymerase II (pol II)
c. DNA polymerase III (pol III) synthesizes both leading
and lagging strands
K. Prokaryotic DNA Replication: Bi-Directional Replication and Leading
Strand Synthesis
1. As previously stated, bi-directional replication in bacteria
occurs in a similar manner as eukaryotes
a. Both leading strand and lagging strand synthesis
b. Both forks are moving away from the origin of
replication
2. Just like the eukaryotic DNA polymerases, DNA polymerase III
(pol III) requires a free 3’ OH group to be able to bind DNA and
start the synthesis
3. Therefore, an RNA primer must be laid down before pol III can
then mediate synthesis on both leading and lagging strands
4. DnaG (also known as primase) lays down an RNA primer (~1012 nt) long, and this primer will provide the free 3’OH
5. Just like the eukaryotes, the leading strand is synthesized in a
continuous manner
6. The template for the leading strand is the strand that goes
5’3’ away from the replication fork
7. The leading strand is synthesized in the 5’3’ direction
towards the replication fork
8. In prokaryotes, DNA polymerase III will bind the DNA at the
free 3’OH and add nucleotides with nitrogenous bases that are
complementary to the sequence in the template
L. Prokaryotic DNA Replication: Bi-Directional Replication and Lagging
Strand Synthesis
1. Just like in eukaryotic replication, lagging strand replication in
prokaryotes is also discontinuous
2. In prokaryotic replication, lagging strand synthesis is done by
producing Okazaki fragments
3. Since lagging strand synthesis is discontinuous, a primers must
be regularly laid down
4. In order to do this, at the beginning of replication DnaG binds
DNA helicase, and moves along the lagging strand template with
DNA helicase, laying down RNA primers as the DNA becomes
single stranded
5. pol III will then bind the DNA at the free 3’OH and add
nucleotide to synthesize new DNA
6. Now that we’ve synthesized the Okazaki fragments, we must
remove the RNA primers, and fill in the sequence
7. On the lagging strand, DNA polymerase III will synthesize DNA
until it reaches the next Okazaki fragment
8. DNA polymerase I (pol I) has 5’3’ exonuclease activity and
polymerase activity
9. DNA polymerase I will then remove the RNA primer and fill in
the sequence using the final 3’OH group of the upstream Okazaki
fragment
10. After DNA polymerase I has replaced the last nucleotide, only
a nick remains
11. This nick is due to the fact that the 3’ OH last nucleotide
added by DNA polymerase I has not been joined to the 5’ PO4 of
the first DNA nucleotide in the next fragment
12. To join the 3’ OH and the 5’ PO4, DNA ligase is used
M. Prokaryotic DNA Synthesis: Termination of Bi-Directional DNA
Synthesis
1. Unlike Eukaryotic DNA replication, the mechanism of how DNA
replication is terminated in prokaryotes is much better
understood
2. As DNA synthesis occurs, the two replication forks will move
around the circular chromosome
3. When these replication forks meet, replication then
terminates, and we have two new DNA molecules
4. Termination of replication occurs when the two forks meet
each other on the opposite side of the chromosome from the OriC
5. Termination occurs at termination sites (Ter A-F)
6. Each replication fork as it moves around the circular
chromosome moves a different speeds
7. The termination sites are bound by a protein Tus, which plays
a role in halting the replication forks
a. Leading strand synthesis arrests at the point of contact
with Tus
b. The final lagging strand RNA primer sites are 50-70 nt
away from Tus
8. Based on the speed of how the replication forks are moving
three possible scenarios for termination exist
9. Scenario 1: If the clockwise moving fork is moving faster and
reaches Ter F,B,C, it will stop
a. Somehow the clockwise moving fork will get through the
TerA, TerD and TerE
b. The clockwise fork will wait for the counter-clockwise
fork to meet it
10. Scenario 2: If the counter-clockwise is moving faster and
then reaches Ter A, D, E, it will stop
a. Somehow the counter-clockwise moving fork will get
through the Ter F, TerB, TerC sites
b. The counter-clockwise fork will wait for the clockwise
moving fork
c. Scenario 3: Replication forks are moving at close to
equivalent speeds and can meet between the Ter A,D,E
cluster and the Ter F,B,C cluster
N. Prokaryotic DNA Replication: Resolving The Two DNA Molecules at
the end of Bi-Directional DNA replication
1. Since prokaryotic chromosomes are circular (unlike in
eukaryotes), near the end of replication, the new chromosomes
are entwined as two interlocking rings (catenae)
2. Chromosomes need to be unlinked through a process called
decatenation before they move to the two new daughter cells
3. Denaturation of the remaining double helix will leave some
single stranded DNA where the replication forks meet
4. DNA repair synthesis will fill in the gaps where the DNA is
single stranded