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D loops
(mitochondria and chloroplast DNA)
H strand is used
as template DNA
L strand begins
to function as
template DNA
RNA polymerase
transcribes a primer,
whose 3’ end is
generated by cleavage by
an endonuclease at
several discrete sites.
The endonuclease is
specific for the triple
structure of DNA-RNA
hybrid.
The 3’ end is then
extended by the DNA
polymerase.
D loop expansion
continues until it
reaches a point
about 2/3 of the way
around the circle.
Replication of this
region exposes an origin
in the displaced L
strand. Synthesis of an
H strand initiates at
this site, which is used
by a special primase
that synthesizes a short
RNA. The RNA is then
extended by DNA
polymerase, proceeding
around the displaced
single-stranded L
template in the opposite
direction from L-strand
synthesis.
Rolling circle
Replicative form
Circular duplex DNA (RF)
Initiation occurs on one strand. The A
protein, nicks the (+) strand of the
duplex DNA at a specific site that
defines the origin for replication.
Elongation of growing strand
displaces old strand
Continued elongation generates
displaced strand of multiple unit
lengths
(ssDNA)
(dsDNA)
Circular ssDNA is
packaged into the phage
virion (M13, f1, 
X174). Upon infection
single-stranded DNA is
converted to doublestranded DNA (RF)
It may be used as a
rolling circle to generate
more progeny
Plasmid ColE1
5’
The sequence of RNA I is
complementary to the 5’
region of primer RNA
origin
5’
3’-OH
5’
5’
Proposed model
No base pairing between
RNA I and RNA primer
DNA
replication
RNase H
Rop protein
stabilizes
RNA
structure
Base pairing with
RNA I may change
the secondary
structure of the
primer RNA
No DNA
replication
The problem
of linear
replicons
- The problem may be
circumvented by converting the
linear replicon into a circular or
multimeric molecule (T4 and
lambda phages).
-The DNA may form an unusual structure (hairpin).
- Instead of being precisely determined, the end may be variable (eukaryotic
chromosomes).
- The most direct solution is for a protein to intervene to make initiation possible
at the actual terminus.
Adenovirus
55 K daltons protein
Initiation of DNA replication
In several viruses that use such mechanisms, a protein is found covalently
attached to each 5’end. In the case of adenovirus, a terminal protein is linked to
the mature viral DNA via a phosphodiester bond to serine.
Telomeric DNA
The telomerase
extends the
telomeric DNA
at the 3’-end
Preventing the ends of a
linear dsDNA molecule
from gradually getting
shorter during successive
rounds of DNA replication.
The telomerase is a
reverse transcriptase
Completation of the extension process at the
end of a chromosome
Telomerase
binding
proteins
Telomerase was found in abundance
in cancer cells indicating that such
enzyme is essential for cancer cells to
continue dividing. Most cells in
human undergo a limited number of
cell divisions and then cease
dividing. In these cells telomerase is
basically not present.
It is believed that after telomerase has
extended the 3’-end a sufficient
amount, DNA pol. a primes and DNA
pol. d synthesizes a new Okazaki
fragment.
Structure of origins of replication
DnaA
T antigen
ORC
The DNA sequences of replicators
share two common features:
-Binding sites (green) for initiation
proteins that nucleates the assembly of
replication machinery
- A stretch of A-T rich DNA that
unwinds readily but not spontaneously
(blue)
Strategy for the
identification of origins of
replication in E. coli
(Factor Accumulation model)
In addition:
* over-expression of DnaA leads
to a burst of initiation.
* under-expression of DnaA leads
to increased initiation mass.
v
E. coli replication origin oriC
Mutants with a defect in any one of the genes for the main histone-like
proteins (HU, FIS, H-NS, StpA, IHF) are viable. Even deficiency in
two histone-like proteins is possible.
Mutations in fis or him (IHF) genes result in asynchronous initiations.
Cells depleted of HU, IHF and H-NS simultaneously are not viable.
Model for initiation complex at oriC
A compact complex is formed in the presence of HU protein and ATP which
contains about 10 monomers of DnaA per oriC. The result of DnaA binding is
that double helix melts within the 3 AT-rich, 13-nucleotides repeats.
Base changes in individual DnaA boxes have surprisingly little
effects. Any change of distances, however, inactivates oriC.
For bacteria dividing more frequently than every 60 minutes, a cycle of
replication must be initiated before the end of the preceding division
cycle.
At division (35/0 minutes), the
cell receives a partially
replicated chromosome.
At 10 minutes, when this "old"
replication fork has not yet
reached the terminus, initiation
occurs at both origins on the
partially replicated
chromosome. The start of
these "new" replication forks
creates a multiforked
chromosome.
Cells growing more rapidly are larger and possess a greater number of
origins and there are correspondingly more chromosomes in the
individual bacterium.
Una cellula viene esposta per
un breve tempo a [3H] timina
(fase di pulse), poi viene
somministrato un eccesso di
timina fredda (fase di chase).
Termination of DNA replication in E. coli
The termination sequences function
in only one orientation. This
arrangement creates a "replication
fork trap;" if for some reason one
fork is delayed, so that the forks fail
to meet at the usual central position,
the more rapid fork will be trapped
at the ter region to wait for the
arrival of the slow fork.
Termination requires the product of
the tus gene, which codes for a
protein (36 kDa) that recognizes the
consensus sequence and prevents the
replication fork from proceeding.
Tus binds to the consensus sequence, where it provides a contrahelicase activity and stops DnaB from unwinding DNA. A difficulty in
understanding the function of the system in vivo is that it appears to be
dispensable, since mutations in the ter sites or in tus are not lethal.
Replication of eukaryotic DNA
Model for DNA replication
?
DNA polymerases d and e are
the main replicative enzymes
Switching delle
Polimerasi
Il prodotto del complesso
DNA polimerasi/α primasi
è di circa 50-100
nucleotidi.
Le DNA polimerasi d and
e sintetizzano
rispettivamente il
filamento guida e quello
copia.
The combined DNA polymerase and
primase activities of the DNA
polymerase a may constitute an
evidence that this enzyme synthesizes
the Okazaky fragments (?) but the
DNA polymerase a has not the 3’
5’ exonuclease activity.
None of eukaryotic DNA Pol. have
the 5’ 3’ exonuclease activity like
Pol. I in E. coli.
Isolating the origins of yeast replicons (ARSs)
S. cerevisiae mutants can be "transformed" to the wild phenotype by addition
of DNA vectors that carries a wild-type copy of the gene (selection).
Some yeast DNA fragments (when circularized) are able to transform
defective cells very efficiently. These fragments can survive in the cell in the
unintegrated (autonomous) state, that is, as self-replicating plasmids. A highfrequency transforming fragment possesses a sequence that confers the ability
to replicate efficiently in yeast. This segment is called an ARS (autonomously
replicating sequence).
Yeast origin of replication
The subdomain B2
appears to correspond
with the 13-nucleotide
repeat array of E.coli.
200 bp
The subdomain B3 is
the DNA binding site for
ABF1 that by
introducing a torsional
stress, favours DNA
melting at B2.
core region (40 bp)
ORCs appear to remain
attached to the yeast
origins throughout the
cell cycle (it is not the
initiator proteins) and
may have a key role in
regulation of DNA
replication.
origin recognition
complex
ARS binding
factor 1
Priming in eukaryotes
RPA (replication protein A)
prevents duplex formation.
3’
5’

10 bases
RNA
In eukaryotes the Okazaki fragments
are much shorter (200 bp) than in
bacteria (about 4000 fragments of 1-2
Kb) and priming is a highly repetitive
event. This indicates that each round
of synthesis replicates the DNA
associates with a single nucleosome
(140-150 bp wound around the core
particle plus 50-70 bp of linker).
DNA (50-100 bases)
DNA pol. a
In eukaryotes the
primase is an integral
part of DNA pol. a.

DNA pol. d
Possibly DNA synthesis (leading and
lagging strands) involves the combined
action of both DNA polymerases d and e
Formazione del complesso pre-replicativo
Cdc6 
Replicazione del DNA in assenza di mitosi
Cdc6 
Assenza dei complessi of pre-replicativi
La proteina Cdc6 è stata inizialmente identificata nel lievito, poi negli
eucarioti più complessi.
Origin
Recognition
Complex
ORC lega l’origine.
Cdc6 è una proteina altamente
instabile (half-life <5 minuti) e
viene rapidamente degradata.
Helicase
ORC recluta Cdc6 che a sua volta
favorisce l’attacco di d 2 copie
dell’elicasi (Mcm) legata alla
proteina Cdt1.
L’idrolisi dell’ATP determina il
distacco delle proteine Cdc6 e
Cdt1.
Complesso Pre-Replicativo
Complesso Pre-Replicativo si
forma nella fase G1 del ciclo
cellulare.
Attivazione del complesso pre-replicativo
ed assemblaggio del replisoma eucariotico
Le Cdk and Ddk sono inattive in
G1 e sono attivate quando le
cellule entrano nella fase S.
Le proteine Cdc6 e Cdt1 si
dissociano dal complesso e sono
rapidamernte degradate durante
la fase S. In tal modo queste
proteine non sono più disponibili
per il ricaricamento di Mcm..
Vanno a costituire
insieme a Mcm2-7 il
complesso CMG
Proteine accessorie
DDK fosforila Mcm mentre
CDK fosforila le proteine
accessorie SId2 e SId3.
SId2, SId3 e Dpb11
favoriscono il legame di
Cdc45 e GINS che stimolano
l’attività ATPasica ed elicasica
di Mcm.
Mentre Mcm è legata al DNA a doppio filamento (dsDNA) il complesso attivo
CMG (Cdt45/ Mcm/GINS) lega il DNA a singolo filamento (ssDNA).
La DNA Pol ε si associa all’origine per prima insieme a Cdt45 quando il DNA è
a doppio filamento.
Le DNA Pol α/primasi ed δ si associano successivamente quando il DNA è a
singolo filamento.
Centinaia o addirittura
migliaia di origini
devono essere attivate
una sola volta in ciascun
ciclo.
Le CDK sono richieste
per attivare il pre-RC.
Una elevata attività CDK
promuove l’inizio della
replicazione del DNA.
L’attività delle CDK
inibisce la formazione di
nuovi complessi pre-RC
I livelli di CDK variano durante il ciclo cellulare
Gli elevati livelli di
Cdk determinano la
fosforilazione e
conseguente
dissociazione
dall’origine di
replicazione delle
proteine
Gli elevati livelli di CDK inibiscono l’attività
di ORC, Cdc6 e Cdt1.
Completation of lagging strand synthesis in eukaryotes
None of the eukaryotic
polymerase have a 5’ 3’
exonuclease activity.
FEN1 endonuclease cannot
initiate primer degradation
because its activity is
blocked by the
triphosphate group present
at the 5’-end of the primer.
The flap model
The RNase H model
The Pol δ/PCNA
DNA-RNA
junction
The Pol δ/PCNA complex drives the gap
filling and formation of the flap
structure in Okazaki fragment.