Alternative modes of circular DNA replication
Rolling circle replication
Rolling circle replication occurs in the multiplication of
many bacterial and eukaryotic viral DNAs, plasmid
replication , and in certain cases of gene amplification.
A phosphodiester bond is broken in one of the strands of a
circular DNA.
Synthesis of a new circular strand occurs by addition of
dNTPs to the 3′ end.
Alternative modes of circular DNA replication
Rolling circle replication
For many plasmid in bacteria cells, replication
is not tied to chromosomal replication.
Many plasmid replicate autonomously by a
method called rolling-circle replication.
A replication initiator protein, called RepA is
encoded by a plasmid gene.
RepA binds to a section of the double
stranded DNA , called the origin of replication,
or oriC.
RepA nicks one strand of the DNA and hold
onto the 5’ end of the strand. The 3’ end,
with its free OH group serves as a primer
for a host DNA polymeraes to begin to
replicate the intact complementary strand.
Alternative modes of circular DNA replication
Rolling circle replication
The RepA recruits a helicase that unwinds the DNA. As the DNA
unwinds, it becomes coated by single-strand DNA binding-protein.
As the replication proceeds, the new single strand, which continues
to be covered with SSB, progressively peels off until replication is
complete.
Alternative modes of circular DNA replication
Rolling circle replication
The two ends of the nicked single strand are rejoined by the RepA
protein and release. DNA ligase seals the nick in the double stranded
molecule
Alternative modes of circular DNA replication
Rolling circle replication
The single stranded DNA can now be replicated.
A region of the DNA becomes looped, allowing RNA polymerase access
to the DNA to form a primer.
Host DNA polymerases use the primer as a starting point for the
synthesis of DNA. Finally DNA ligase seals the remaining nick,
resulting in a double stranded plasmid
Models for organelle DNA replication
There is no consensus on the mode of replication of organelle DNA.
The various models proposed for mitochondrial DNA (mtDNA) and
chloroplast DNA (cpDNA) replication remain controversial.
Models for chloroplast DNA (cpDNA) replication
• A subject of debate particularly since there is controversy over
whether cpDNA is linear or circular.
• Some evidence for a strand displacement model.
• Other models include a theta replication intermediate, rolling
circle replication, and recombination-dependent replication.
Models for mitochondrial DNA (mtDNA) replication
• DNA polymerase γ (gamma) is used exclusively for mtDNA
replication.
• Two models for replication have been proposed: the strand
displacement model and the strand coupled model
Models for organelle DNA replication
Strand displacement model:
The most widely accepted model for mammalian mtDNA.
Replication is unidirectional around the circle and there is one
replication fork for each strand (light strand (L) and heavy strand
(H)).
There is one priming event per template strand – thus, there are two
origins (OH and OL).
Models for organelle DNA replication
Strand displacement model:
Replication begins in a region called the “D” loop – a region of 500–600 nt.
A preformed RNA primer (synthesized by the mitochondrial RNA polymerase) is
required at both origins. The H strand is used as a template first to make a
new L strand, starting at OH.
Models for organelle DNA replication
Strand displacement model:
After approximately two-thirds of the mtDNA has been copied by DNA
polymerase, the replication fork passes the major origin of L strand synthesis.
Only after the displaced H strand passes the origin on the L strand, synthesis
of a new H strand start from OL.
Synthesis is continuous around the circle on both strands. The RNA primers are
cleaved by the multifunction endoribonuclease RNase MRP
Models for organelle DNA replication
Strand coupled model:
In this model the coupled leading (H
strand) and lagging strand synthesis
represents a semidiscontinous,
bidirectional mode of DNA replication.
Synthesis of Okazaki fragments on the
lagging strand.
Genomes
In each organism (from bacteria to human) very long
molecules of DNA must be intricately folded so they can fit
into the cell.
DNA is associated with architectural proteins and
packaged into chromosomes.
But, genetic information has to be accessible for processes
such as replication and transcription.
THUS
Interconnection between architectural protein and DNA has
to be flexible enough to switch between folding and opening
up state.
Genomes size in different organism
Packaging of the eukaryotic genome
Packaging of the eukaryotic genome
The problem:
How to fit 2 meters of DNA into the spherical nucleus (a <10
µm space) ?
The solution:
Each DNA molecule is wrapped around histone proteins to form
nucleosomes (chromatin), which are then condensed to make up
an entire chromosome.
DNA is compacted 10000 fold respect to naked DNA
Most eukaryotes package their genomes with histones.
There are some exceptions:
• Dinoflagellates package their DNA with small basic non-histone
proteins.
• Sperm DNA is compacted with basic proteins known as
protamines.
THE NUCLEOSOME
Nucleosome are the building blocks of chromosomes
The majority of the DNA of the eukaryotic cells is packaged into NUCLEOSOME.
Each nucleosome is composed of a core of eight histone proteins and the DNA
wrapped around them. The DNA mostly tightly associated with the nucleosome is
called core DNA and the
length of DNA associated with each nucleosome is 147 bp.
By assembling into nucleosomes, the DNA is compacted approximately six fold.
The DNA between each nucleosome is called linker DNA.
The length of the linker DNA is variable.
Histones are small, positively charged proteins
Eukaryotic cells commonly contain five abundant histones:
H1, H2A, H2B, H3 and H4.
Histones H2A, H2B, H3 and H4 are the core histones, and two
copies of each of these histones form the protein core (octamer)
around which nucleosomal DNA is wrapped.
Histones have a high content of positively
charged amino acids in particular lysine
or arginine (20%)
Within octamer, two H3/H4 dimers associate to form a tetramer,
while the two H2A/H2B dimers associate at each end of the
H3/H4 tetramer in presence of DNA.
Histones are small, positively charged proteins
All four core histones contain a
region that interact with other
histones and DNA (Histonesfold domain). This region allow
histones to come together like
“clasping hands”.
The core histones also have an
amino-terminal extensions,
called “tails” that stabilize DNA
wrapping around the octamer.
The packaging of DNA: the 30 nm fiber
The packaging of DNA: the 30 nm fiber
Once nucleosomes are formed, the next step in the packaging of
DNA is the binding of histone H1 (linker histone).
Histone H1 in not part of the nucleosome core particles.
H1 is a small positively charged protein that interacts with linker
DNA between nucleosomes, further tightening the association of
the DNA with nucleosomes.
H1 binding produce a 30-nm fiber.
There are two model for the 30-nm
fiber.
The packaging of DNA: the 30 nm fiber
Solenoid model
The nucleosomal DNA forms a superhelix containing
approximately six nucleosomes per turn.
This model has been accepted for many years, however, it turns
out that solenoids are not seen at physiological salt
concentration.
The packaging of DNA: the 30 nm fiber
Zigzag model
Recently studies suggest that, at least in transcriptionally
active cells, nucleosomes do not form solenoid. Instead they
adopt a zigzag structure in which the linker DNA pass through
the central axis of the fiber.
Further packing of DNA involves loop domains
The 30-nm fiber is further compacted into structures not yet fully
understood. One popular model proposes that the 30-nm fiber forms
loops of 40-90 kb that are held together at their bases by a protein
structure referred to as the nuclear scaffold.
Two classes of protein that contribute to the nuclear scaffold has
been identified. Topoisomerase II and the SMC (structural
maintenance of chromosome) proteins hold the DNA at the base of
each loop keeping it isolate from one another.
Fully condensed chromatin:
packing ratio of 10.000 fold
DNA replication in
eukaryotes
Eukaryotic origins of replication
• Bidirectional replication starts not at the end but at many
internal sites on linear chromosomes.
• Mice have 25,000 origins, spanning ~150 kb each.
• Humans have 10,000 to 100,000 origins.
• In the yeast Saccharomyces cerevisiae there is a consensus
sequence called an autonomous replicating sequence (ARS).
• Mammalian origin sequences are usually AT rich but lack a
consensus sequence.
Selective activation of origins of replication
• The overall rate of replication is largely determined by the number of
origins used and the rate at which they initiate.
• During early embryogenesis, origins are uniformly activated.
• Later in the development, cell division slows down and zygotic gene
expression begins. At this stage, replication becomes restricted to
specific origin sites.
• The parameters of this selective activation are no clear but may
include changes in nucleotide pool or changes in chromatin structure.
• Replication forks in mammalian cell are
not distributed diffusely throughout
the nucleus. Replication forks are
clustered in subnuclear compartments
called “replication factories.”
• Forty to many hundreds of forks are
active in each factory.
1- Histone removal at origins of replication
The process of DNA replication in eukaryotes involves 12 major steps.
In the first step, histone removal at the origin of replication allows
access of the replication machinery to the template DNA. How this is
achieved has yet to be determined.
Histone modifications (particularly acetylation) and chromatin
remodeling factors may loosen the chromatin to allow disassembly of
the nucleosomes and access to the template DNA.
Prereplication complex formation and replication
licensing
All organism must replicate DNA
before cell division.
DNA synthesis is restricted to a
specific phase of the cell cycle (S
phase)
One major difference between bacterial and eukaryotic
DNA replication is that in bacterial cells, as soon as the
initiator proteins accumulate at the origin, DNA helicases
are recruited to the origin and initiation begins.
In contrast, eukaryotic cells separate origin selection from
initiation, through the formation of a pre-replication
complex (PR-C). Separation of these two events prevents
over-replication of the genome
2- PrePre-replication complex formation (Pre
(Pre--RC)
RC)
Once eukaryotic chromatin has been open up, the second step is prereplication complex formation (pre-RC) at the origin of replication.
The ATP-dependent origin recognition complex (ORC) binds origin
sequences. ORC composed of six polypeptide subunits (Orc1–6).
3- Replication licensing
Once bound , ORC recruits at least 2 additional proteins, Cdc6 and Cdt1 (helicase
loading protein ). These three proteins use ATP binding and hydrolysis to load the
ring shaped Mcm2-7 helicase complex around DNA to complete the formation of
the pre-RC. Mcm2-7 is a hexameric complex.
Cell cycle regulate this step to ensure that DNA only replicates once per cell cycle.
Regulation of the replication licensing system by
CDKs
Cell cycle
Regulation of the replication licensing system by
CDKs
Replication licensing is regulated by
the activity levels of cyclindependent kinases (CDKs). Cyclindependent kinases are key
activators of the cell cycle
transitions.
When CDK activity is low in the G1
phase, Mcm2-7 can be loaded into
origins to prepare for replication.
When CDK activity is high in the S
and G2 phases no further Mcm2-7
can be loaded into origin.
Because CDK activity remain high
until the end of mitosis, no new
Mcm2-7 can be loaded until
chromosome segregation is
complete.
4- Duplex unwinding and relaxing of supercoils
Cdc6 and Cdt1 are released from the complex, and other replication
factors are recruited (the single stranded DNA binding protein, called
replication protein A RPA).
RPA protects the single stranded DNA from nuclease attack.
The helicase activity of Mcm2-7 unwinds the DNA duplex.
Topoisomerase I and or II resolve positive supercoils ahead of the
replication fork.
DNA polymerases in eukaryotes
Eukaryotic cells also have multiple DNA polymerases (more than 15).
Of these, three are essential to duplicate the genome.
• DNA Pol α/primase, synthesizes
an RNA primer and is rapidly
replace by the highly processive
DNA Pol δ or Pol ε. (Polymerase
switching)
• DNA Pol ε is specialized to
synthesize the leading strand
and DNA Pol δ the lagging
strand.
• As in bacterial cells the majority
of the remaining eukaryotic DNA
polymerases are involved in DNA
repair.
5- RNA priming of leading and lagging strand
Once present at the origin, DNA polymerase α/primase synthesizes
an RNA primer and briefly extend it
6- Polymerases switching
The resulting primer-template junction is recognized by the sliding clamp
loader (RFC), which assembles a sliding clamp (PCNA) at these sites.
PCNA is a ring-shaped trimer. In the presence of ATP, the clamp loader
RFC opens the trimer and passes DNA into the ring and then reseals it.
Either DNA Pol δ or DNA Pol ε recognized this primer and begins leading
and lagging strand synthesis, respectively
7- Elongation of leading and lagging
strand
8- Continuous synthesis on the
leading strand; polymerase switching
on the lagging strand
9- Removal of RNA primer
Two different pathways proposed for
RNA primer removal:
1 - Ribonuclease H1 nicks the RNA
primer and the primer is degraded by
FEN-1 (flap endonuclease 1)
2 - DNA pol δ causes strand
displacement and FEN-1 removes the
entire RNA containing 5′ flap.
FEN-1 is a structure-specific 5′ nuclease
with both exonuclease and endonuclease
activity.
PCNA-coordinated rotary handoff
mechanism of DNA from DNA pol ε to
FEN-1.
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- Fill in of gaps left by primer removal
The remaining gaps left by primer
removal are filled in by DNA polymerase
δ or ε.
End product is a nicked double-stranded
DNA.
Nicks are sealed by DNA ligase I.
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- Joining of the Okazaki fragments
In association with PCNA, DNA ligase
I joins the Okazaki fragments by
catalyzing the formation of new
phosphodiester bonds.
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- Histone deposition
Nucleosomes re-form within approximately
250 bp behind the replication fork.
Chromatin assembly factor 1 (CAF-1)
brings histones to the DNA replication fork
in association with PCNA.
Histones H3 and H4 form a complex and are
deposited first, followed by two histone
H2A-H2B dimers.
In eukaryotes, replication continues until one
fork meets a fork from the adjacent
replicon.
The progeny DNA molecules remain
intertwined.
Toposiomerase II is required to resolve the
two separate progeny genomes.