REPLICAREA, REPARAREA I

RECOMBINAREA ADN
Prof. Dr. Marieta COSTACHE

Replicarea ADN este semi-conservativ

O molecul de ADN este
duplicat
(replicat)
prin
polimerizarea unei noi catene
complementare pe fiecare din
vechile catene ale dublei helice de
ADN.
Procesul de replicare a
ADN, n care dou molecule de
ADN identice sunt formate
plecnd de la molecula de
origine, permite copierea i
transmiterea informaiei genetice
de la o celul la celulele fiice i de
la prini la descendenii lor ;

Experiment
MeselsonStahl

Experimentul MeselsonStahl

The Meselson-Stahl experiment

(a) Cells were grown for many generations in a
medium containing only heavy nitrogen, 15N,
so that all the nitrogen in their DNA was 15N,
as shown by a single band (blue) when
centrifuged in a CsCl density gradient;
(b) Once the cells had been transferred to a
medium containing only light nitrogen, 14N,
cellular DNA isolated after one generation
equilibrated at a higher position in the density
gradient (purple band), (c) Continuation of
replication for a second generation yielded two
hybrid DNAs and two light DNAs (red),
confirming semiconservative replication.

Majoritatea proceselor de replicare a ADN sunt

bidirecionale

Replicarea (continuare)
n momentul replicrii unei
molecule de ADN, catenele sale sunt
deprtate pentru a forma una sau
mai multe furci de replicare n
form de Y.
Enzima ADN
polimeraza care se poziioneaz la
nivelul
fiecrei
furci
este
responsabil de sinteza noii catene
de ADN complementar cu fiecare
din catenele parentale, sintetiznd
astfel dou molecule de dublu
helix ;

Majoritatea
replicrii ADN este
bidirecional

Demonstrarea creterii
bidirecionale
prin
autoradiografie: dac celulele
replicative n cultur sunt
expuse unei concentraii mari
de timidin [3H], ADN obinut
va fi puternic marcat (hot) la
nivelul originii de replicare i
mai slab n apropierea acesteia
(warm). Prin autoradiografie
au fost obinute profilele b care
confirm ipoteza din figura a.

Replicarea
la
Drosophila

Schematic diagram of leading-strand and lagging-strand DNA synthesis at a replication fork.

Nucleotides are added by a DNA polymerase to each growing daughter strand in the 5n 3direction (indicated by arrowheads).
The leading strand is synthesized continuously from a single RNA primer (red) at its 5end. The lagging strand is synthesized discontinuously
from multiple RNA primers that are formed periodically as each new region of the parental duplex is unwound. Elongation of these primers
initially produces Okazaki fragments. As each growing fragment approaches the previous primer, the primer is removed and the fragments are
ligated. Repetition of this process eventually results in synthesis of the entire lagging strand.

Replicarea ADN ncepe la nivelul unor situsuri

cromozomiale specifice numite origini de replicare
Secvene consens minimale de la nivelul originii de replicare la bacterii

In funcie de organism originile de replicare sunt: (1) segmente

de ADN unice cu mai multe uniti repetitive scurte; (2) recunoscute de
proteine multimere origin-binding proteins; (3) de obicei conin
secvene bogate n A-T

Probleme pe care trebuie s le rezolve

polimerazele pentru a realiza copierea ADN
ADN polimerazele nu sunt capabile s nlture legturile de
hidrogen pentru a separa cele dou catene care trebuiesc copiate
Toate polimerazele cunoscute pot numai elonga o caten
preexistent de ADN sau (primer-ul) i sunt incapabile s iniieze
sintez de novo
Cele dou catene ale ADN sunt complementare i antiparalele, dar toate polimerazele nu pot cataliza dect reacia de
adugare de nucleotide numai la captul 3-hidroxil al catenei n
formare, deci lanul crete numai n direcia 5 - 3.
Numai una dintre catenele furcii de replicare, catena precoce,
poate fi sintetizat continuu. Cealalta caten ADN, denumit
ntrziat poate fi sintetizat de ctre polimeraz, discontinuu, sub
forma unor fragmente scurte de ADN care sunt ulterior legate cu
ajutorul ADN ligazei pentru a da natere unui singure catene
continue.

Replicarea la E. Coli este iniiat de ctre

proteina DnaA

La E. coli DnaB helicaza este responsabil

de desfacerea duplexului ADN

La nivelul furcii de replicare una dintre catene

este replicat continuu i cealalt discontinuu
pornind de la mai muli primeri

ADN polimerazele de la E. coli

Sinteza la
nivelul
catenei
ntrziate

Model for initiation of

replication at the E coli
origin, oriC.

Arrangement of sequences in the E. coli replication origin,

oriC. Although the repeated sequences (shaded in colour) are
not identical, certain nucleotides are particularly common in
each position, forming a consensus sequence. In positions
where there is no consensus, N represents any of the four
nucleotides. The arrows indicate the orientations of the
nucleotide sequences.

Model for initiation of replication at the E coli origin, oriC.

(I) About 20 DnaA protein molecules, each with a bound ATP, bind at
the four 9 bp repeats. The DNA is wrapped around this complex.
2) The three A=T-rich 13 bp repeats are denatured sequentially.
3) Hexamers of the DnaB protein bind to each strand, with the aid of
DnaC protein. The DnaB helicase activity further ununwinds the DNA
in preparation for priming and DNA synthesis.

Correctly paired
bases are required
for DNA polymerase
catalyzed nucleotide
addition

Correctly paired bases are required for DNA polymerase catalyzed nucleotide
addition. a) Schematic diagram of the attack of a primer; 3'OH end on a correctly base-paired
dNTP.
b) Schematic diagram of the consequence of incorrect base-pairing on catalysis by DNA
polymerase. In the example shown, the incorrect A% base pair displaces the a-phosphate of the
incoming nudeotide. This incorrect alignment reduces the rate of catalysis dramatically resulting
in the DNA polymerase preferentially adding correctly base-paired dNTPs.

Schematic illustration of
the steric constraints
preventing catalysis
using rNTPs by
DNA polymerase.
Schematic illustration of the
steric constraints preventing
catalysis using rNTPs by DNA
polymerase. a) Binding of a
correctly base-paired dNTP to
the DNA polymerase. Under
these conditions, the 3"OH of the
primer and the (x- phosphate of
the dNTP are in close proximity.
b) Addition of a 2'OH results in a
steric dash with amino acids (the
discriminator amino acids) in
the nudeotide binding pocket.
This results in the c -phosphate
of the dNTP being displaced and
a misalignment with the 3'OH of
the
primer,
dramatically
reducing the rate of catalysis

Elongation of a
DNA chain

An example of error correction by the 3- 5' exonuclease

activity of DNA polymerase I

An example of error correction by the 3'^5' exonuclease activity of DNA polymerase I. Structural analysis has located the exonuclease
activity ahead of the polymerase activity as the enzyme is oriented in its movement along the DNA. A mismatched base (here, a C-A
mismatch) impedes translocation of DNA polymerase I to the next site. Sliding backward, the enzyme corrects the mistake with its
3'-5' exonuclease activity, then resumes its polymerase activity in the 5'>3' direction.

Nick translation

In this process, an RNA or DNA strand paired to a

DNA template is simultaneously degraded by the
5'>3' exonuclease activity of DNA polymerase I
and replaced by the polymerase activity of the same
enzyme. These activities have a role in both DNA
repair and the removal of RNA primers during
replication (both described later). The strand of
nucleic acid to be removed (either DNA or RNA) is
shown in green, the replacement strand in red. DNA
synthesis begins at a nick (a broken phosphodiester
bond, leaving a free 3' hydroxyl and a free 5'
phosphate). Polymerase I extends the nontemplate
DNA strand and moves the nick along the DNA a
process called nick translation. A nick remains
where DNA polymerase I dissociates, and is later
sealed by another enzyme.

Synthesis of Okazaki fragments

Synthesis of Okazaki fragments

(a) At intervals, primase synthesizes an
RNA primer for a new
Okazaki
fragment. Note that if we consider the
two template strands as lying side by
side, lagging strand synthesis formally
proceeds in the opposite direction from
fork movement.
(b) Each primer is extended by DNA
polymerase III. (c) DNA synthesis
continues until the fragment extends as
far as the primer of the previously added
Okazaki fragment. A new primer is
synthesized near the replication fork to
begin the process again.

Fragmente catenei ntrziate sunt ligate

pentru a forma un lan continuu de ADN

Mechanism of the DNA ligase reaction

Mechanism of the DNA ligase reaction. In each of the three steps, one phosphodiester bond is formed at the expense
of another. Steps CD and lead to activation of the 5' phosphate in the nick. An AMP group is transferred first to a
Lys residue on the enzyme and then to the 5' phosphate in the nick. In step , the 3'- hydroxyl group attacks this
phosphate and displaces AMP, producing a O Ribose Adenine phosphodiester bond to seal the nick. In the E.
coli DNA ligase reaction, AMP is derived from NAD+. The DNA ligases isolated from a number of viral and
eukaryotic sources use ATP rather than NAD+, and they release pyrophosphate rather than nicotinamide
mononucleotide (NMN) in step .

Proteins at the E. coli Replication Fork

Subunitatile DNA polimerazei III

DNA synthesis on the leading

and lagging strands
DNA synthesis on the leading
and lagging strands. Events at
the replication fork
are
coordinated by a single DNA
polymerase III dimer, in an
integrated complex with DnaB
helicase. This figure shows the
replication
process
already
underway (parts (a) through (e)
are discussed in the text). The
lagging strand is looped so that
DNA synthesis proceeds steadily
on both the leading and lagging
strand templates at the same
time. Red arrows indicate the 3'
end of the two new strands and
the direction of DNA synthesis.
Black arrows show the direction
of movement of the parent DNA
through the
complex. An
Okazaki fragment is being
synthesized on the lagging
strand.

Two metal ions bound to DNA polymerase

catalyze nucleotide addition
Two metal ions bound to DNA polymerase catalyze
nucleotide addition.
(a) Illustration of the active site of a DNA polymerase.
The two metal ions (shown in green) are held in place
by interactions with two highly conserved Aspartate
residues. Metal ion A primarily interacts with the
3'OH resulting in reduced association between the O
and the H.
This leaves a nudeophilic 3'0 . Metal ion B interacts
with the diphosphates of the incoming dNTP to
neutralize their negative charge. After catalysis, the
pyrophosphate product is stabilized through similar
interactions with metal ion B (not shown). (b) Three
dimensional structure of the active site metal ions
associated with the DNA polymerase, the 3'OH end of
the primer and the incoming nudeotide. The metal ions
are shown in green and the remaining elements are
shown in the same colours as in Figure 8-5b. The view
of the polymerase shown here is roughly equivalent to
rotating the image shown in Figure- 180 around the
axis of the DNA helix.

Proteins Required to Initiate Replication at the

E. coli Origin

Procesivitatea ADN polimerazelor crete

datorit prezenei subunitii dimere clamp

Sinteza catenei continue i discontinue

este concurent

Unele componente ale furcii de replicare

Termination of chromosome replication in

E. coli
(a) The Ter sequences are positioned on the chromosome in two
clusters with opposite orientations, (b) Replication of the DNA
separating the opposing replication forks leaves the completed
chromosomes joined as catenanes, or topologically interlinked
circles. The circles are not covalently linked, but because they
are interwound and each is covalently closed, they cannot be
separatedexcept by the action of topoisomerases. In E. coli, a
type II topoisomerase known as DNA topoisomerase IV plays
the primary role in the separation of catenated chromosomes,
transiently breaking both DNA strands of one chromosome and
allowing the other chromosome to pass through the break.

Chromosome partitioning
in bacteria

Chromosome partitioning in bacteria

(a) All replication is carried out at a central
replication factory that includes two complete
replication forks.
(b) The two replicated copies of the bacterial
chromosome are extruded from the replication
factory into the two halves of the cell, possibly
with each newly synthesized origin bound
separately to different points on the plasma
membrane. Sequestering the two chromosome
copies in separate cell halves facilitates their
proper segregation at cell division.

Illustration of the path of the template DNA through the

DNA polymerase

Illustration of the path of the template

DNA through the DNA polymerase.
The recently replicated DNA is
associated with the palm region of the
DNA polymerase. At the active site, the
first base of the single-stranded region
of the template is in
a position
expected for double-stranded DNA.
As one follows the template strand
toward its 5' end, the phosphodiester
backbone abruptly bends 90 . This
results in the second and
all
subsequent single-stranded bases being
placed in a position that prevents any
possibility
of base-pairing with a
dNTP bound at the active site.

DNA polymerases
synthesize DNA in a
processive manner

DNA polymerases synthesize DNA

in a processive manner. This
illustration shows the difference
between
a processive and a
nonprocessive DNA polymerase.
Both DNA polymerases bind the
primer: template junction. Upon
bindin 8, the nonprocessive enzyme
adds a single dNTP to the 3" end of
the primer and then is released
from the new primer:template
junction. In contrast, a processive
DNA polymerase adds
many
dNTPs each time it binds to the
template.

DNA helicases separate

the two strands of the double helix

DNA helicases separate the two

strands of the double helix. When
ATP is added to a DNA helicase
bound to ssDNA, the helicase moves
with a defined polarity on the
ssDNA. In the instance illustrated,
the DNA helicase has a 5'---.3'
polarity. This polarity means that
the DNA helicase would be bound to
the lagging strand template at the
replication
fork.

Action of topoisomerase
at the replication fork

Action of topoisomerase at the replication

fork. As positive supercoils accumulate in
front of the replication fork, topoisomerases
rapidly remove them.
In this diagram, the action of Topo II removes
the positive supercoil induced by a
replication fork. By passing one part of the
unreplicated
dsDNA through a doublestranded break in a nearby unreplicated
region, the positive
supercoils can be
removed. It is worth noting that this change
would reduce the linking number by two and
thus would only have to occur once every 20
bp replicated. Although the action of a type
II topoisomerase is illustrated here, type I
topoisomerases can also remove the positive
supercoils generated by the replication fork.

Enzymes that Function at the Replication Fork

Mainria de replicare eucariot este n general

similar cu cea de la E. coli

Rolul ADN polimerazelor

in replicare, reparare si recombinare
Rolul ADN polimerazelor
la eucariote:
in replicare, reparare si recombinare
la eucariote
VIOLETA TRUSCA
Master An II
Biochimie si Biologie Moleculara
Universitatea din Bucuresti
Facultatea de Biologie

45

Principalele ADN polimeraze eucariote:

ADN polimeraza - initierea replicarii ADN

- 4 subunitati distincte:
cea mai mare 180 KDa
cea mai mica ~ 50-60 KDa
- primaza capabila sa sintetizeze mici transcripti de ADN
ce servesc ca primeri pt elongarea catenei ADN,
asigurata de subunitatea catalitica.
< 100 nucleotide / eveniment de legare nu e inalt procesiva

ADN polimeraza - repararea leziunilor ADN

ADN polimeraza - replicarea ADN mitocondrial
ADN polimeraza - replicaza nucleara
in prezenta PCNA (antigenul nuclear din celulele proliferative)

devine mai procesiva (>1000 nucleotide / eveniment de legare)

ADN polimeraza - repararea leziunilor ADN

- activitate exonucleozica 3`-5`
46

ADN polimerazele de la mamifere

Activities and Functions of DNA

Polymerases

ADN polimerazele realizeaza:

SINTEZA REPLICATIVA A ADN:

- implica intreaga molecula de ADN
- realizata in vederea reduplicarii genetice
si continuitatii ereditare.
SINTEZA REPARATORIE A ADN:
- se realizeaza la nivelul unor regiuni scurte
- are loc destramarea catenei afectate
ADN celular poate fi afectat de
mutageni endogeni sau din mediul inconjurator,
rata mutatiei la mamifere
fiind de mai putin de o mutatie / genom / generatie

49

FIDELITATEA ADN POLIMERAZELOR:

Moleculele de ADN intact sunt replicate cu o fidelitate mare
Trei pasi de control:
1. selectia bazelor
2. proofreading
3. repararea ADN imperecheat gresit.
Nucleotidele afectate din ADN pot fi reparate prin sisteme:
- NER (reparare prin excizia nucleotidelor)
- BER (reparare prin excizia bazelor)

50

Erorile de imperechere ~10-3 / pb replicata

- ERORI DE SUBSTITUTIE DE BAZE
incorporarea unei nucleotide nepotrivite
ca urmare a unei imperecheri eronate de baze azotate.
- MUTATII FRAMESHIFT
ca urmare a includerii / omisiunii / deletiei unui nucleotid.
ADN polimerazele realizeaza scrutinul nucleotidelor
ce vin pt a se imperecha cu cele complementare din matrita
controlul erorilor presintetice.

ADN polimerazele pot evalua imperecherea de baze dupa adaugarea

nucleotidelor si inlatura ultima nucleotida gresit adaugata
controlul proofreading (corectia citirii).

51

Familii de ADN polimeraze:

52

53

Modelul ADN polimerazelor din familia A si B:

54

EVOLUTIA DIFERITELOR FAMILII DE ADN

POLIMEREZE:

55

Replication factor C - Rfc

PCNA (Proliferating Cell Nuclear Antigen)

Electron microscopy of replicating SV40 DNA

indicates bidirectional growth of DNA strands from
an origin. The replicating viral DNA from SV40infected cells was cut by the restriction enzyme EcoRI,
which recognizes one site in the circular DNA.
Electron micrographs of treated samples showed a
collection of cut molecules with increasingly longer
replication bubbles, whose centres are a constant
distance from each end of the cut molecules. This
finding is consistent with chain growth in two
directions from a common origin located at the center
of a bubble, as illustrated in the corresponding
diagrams. [See G. C. Fareed et al., 1972, J. Virol.
10:484; photographs courtesy of N. P. Salzman.]

Bidirectional mechanism of DNA replication. The left

replication fork here is comparable to the replication fork
diagrammed in Figure, which also shows proteins other than large
T-antigen. (Top) Two large T-antigen hexameric helicases first
bind at the replication origin in opposite orientations.
Step 1: Using energy provided from ATP hydrolysis, the helicases move in
opposite directions, unwinding the parental DNA and generating singlestrand templates that are bound by RPA proteins.
Step 2: PrimasePol complexes synthesize short primers base-paired to
each of the separated parental strands.
Step 3: PCNA-RfcPol complexes replace the primasePol complexes and
extend the short primers, generating the leading strands (dark green) at
each replication fork.
Step 4 : The helicases further unwind the parental strands, and RPA
proteins bind to the newly exposed single-strand regions.
Step 5: PCNA-RfcPol complexes extend the leading strands further.
Step 6: PrimasePol complexes synthesize primers for lagging-strand
synthesis at each replication fork.
Step 7: PCNA-RfcPol complexes displace the primasePol complexes
and extend the lagging-strand Okazaki fragments (light green), which
eventually are ligated to the 5ends of the leading strands. The position
where ligation occurs is represented by a circle.
Replication continues by further unwinding of the parental strands and
synthesis of leading and lagging strands as in steps 4 7. Although
depicted as individual steps for clarity, unwinding and synthesis of leading
and lagging strands occur concurrently.

Replicarea la drojdii

DNA polymerase
switching during eukaryotic
DNA replication
DNA polymerase switching during
eukaryotic DNA replication. The order
of DNA polymerase function is
illustrated. The length of the DNA
synthesized is shorter than in reality
for
illustrative
purposes.
AlthoTypically the combined DNA Pol
alpha/primase product is between 50100 bp and the further extension by
Pol epsilon or Pol delta is between 100
and 10,000 nucleotides. Both DNA Pol
delta and epsilon can substitute for
DNA Pol alpha/primase, it is likely that
they function in the replication of
specific DNA strands (leading or
lagging). Current studies have yet to
determine which polymerase functions
on which strand, however.

Sliding DNA clamps

increase the processivity
of associated
DNA polymerases.

The composition of the DNA Pol III holoenzyme

The composition of the DNA

Pol III holoenzyme. There are
three enzymes in each copy of
the DNA Pol Ill holoenzyme:
two copies of the DNA Pol III
core enzyme and one copy of
the  complex c. The -complex
includes two copies of the vprotein, each of which includes
a domain that interacts with
one DNA Pol III core. Analysis
of the amino acid sequence of
the -protein indicates that the
DNA Pol III binding region of
the protein is separated from
the part of the protein involved
in clamp loading by an
extended flexible linker.

This linker is proposed to allow

the two
polymerases to move in a relatively independent
manner that would be necessary for one
polymerase to replicate the leading strand and the
other to replicate the lagging strand.

The trombone" model for coordinating replication by two

DNA polymerases at the E. coli replication fork.

(a) The DNA helicase at the E.

coli DNA replication fork travels
on the lagging strand template in
a 5'--->3' direction. The DNA Pol
III holoenzyme interacts with the
DNA helicase through the subunit, which also binds to both
DNA polymerases. One DNA Pol
III core is replicating the leading
strand and the other DNA Pol III
core replicates the lagging
strand. SSB coats the ssDNA
regions of the DNA (for
simplicity SSB on the lagging
strand is only shown in part.

The "rombone" model for coordinating replication by two

DNA polymerases at the E. coli replication fork

(b) Periodically, DNA

primase will associate with
the DNA helicase and
synthesize a new primer
on the lagging strand
template.

The "rombone" model for coordinating replication by two

DNA polymerases at the E. coli replication fork

(c) When the lagging strand

DNA polymerase completes
an Okazaki fragment, it is
released from the sliding
damp and the DNA.

The "rombone" model for coordinating replication by two

DNA polymerases at the E. coli replication fork

d) The recently primed lagging strand

DNA is then a target of the clamp
loader, which assembles a new sliding
clamp at the primer. template junction
created by synthesizing a new RNA
primer.

The "rombone" model for coordinating replication by two

DNA polymerases at the E. coli replication fork
(e) The primer. template junction
with its associated sliding damp binds
to the lagging strand DNA
polymerase, which initiates DNA
synthesis on the next Okazaki
fragment.
Although this description has
concentrated on the more complex
action occurring during the synthesis
of the lagging strand, during this
entire process, new ssDNA template
for the leading strand has been
generated and rapidly replicated by
the leading strand DNA Pol III.

Binding of the DNA helicase to DNA Pol III holoenzyme

stimulates the rate of DNA strand separation

The -subunit of the clamp loader interads with both the DNA helicase and the DNA polymerase at the
replication fork. (a) When this interaction is made, the DNA helicase unwinds the DNA at
approximately the same rate as the DNA polymerases replicate the DNA. (b) If the DNA helicase is not
associated with DNA Pol III holoenzyme, DNA unwinding slows by tenfold. Under these conditions, the
DNA polymerases can replicate faster than the DNA helicase can separate the strands of unreplicated
DNA. This allows the DNA Pol III holoenzyme to "catch up" to the DNA helicase and the reformation
of a full replisome.

SeqAbound
to hemimethylated DNA
inhibits reinitiation from
recently replicated
daughter origins.

a) Prior to DNA replication, GATC

sequences throughout the E. coil genome
are methylated on both strands ("fully"
methylated). Note that throughout the
figure, the methyl groups are represented
by red hexagons. (b) DNA replication
converts
these
sites
to
the
hemimethylated state (only one strand of
the
DNA
is
methylated).
(c)
Hemimethylated GATC sequences are
rapidly bound by SeqA.

SeqAbound
to hemimethylated DNA
inhibits reinitiation from
recently replicated
daughter origins
(d) Bound SeqA protein inhibits the
full methylation of these sequences
and the binding of onC by DnaA
protein (for simplidly, only one of
the daughter molecules is illustrated
in parts d, e, and f). (e) When SeqA
infrequently disassodates from the
GATC sites, the sequences can
become fully methylated by Dam
DNA methyl transferase, preventing
rebinding by SeqA. (f) When the
GATC
sites
become
fully
methylated, DnaA can bind and
direct a new round of replication
from the daughter oriC replicators.

The Replication
Factory Hypothesis

In the left panel, the two DNA

helicases function independently.
In the right panel, the two DNA
helicases remain associated with
one another. Note that in the
right panel, one DNA Pol III
holoenzyme uses only the Watson
strand as a template and the
other uses only the Crick strand
as a template. For simplicity, the
DNA Pol III is not shown
associated
with
the
DNA
helicases.

Chromosome breakage
as a result of incomplete DNA replication

This illustration shows the

consequences of
incomplete
replication
followed
by
chromosome segregation. The top
of each illustration shows the
entire chromosome. The bottom
shows the
details of the
chromosome breakage at the DNA
level.As the
chromosomes are
pulled apart, stress is placed on
the unreplicated DNA, resulting in
the breakage of the chromosome.

Replicators are
inactivated by DNA
replication
A chromosome with five replicators is
shown. The replicators labeled 3 and 5
are the first to be activated, leading to the
formation of two pairs of bidirectional
replication forks. Activation of the
parental replicator results in the
inactivation of the copies of each
replicator on both daughter DNA
molecules until the next cell cycle
(indicated by a red X). Further extension
of the resulting replication forks replicates
the DNA overlapping with the number 2
and 4 replicators. When a replicator is
copied by a fork derived from an adjacent
origin prior to initiation, it is said to have
been passively replicated. Although these
replicators have not initiated, they are
nevertheless inactivated by the act of
replicating their DNA.

In contrast, replicator 1 is not reached by an

adjacent fork prior to initiation and is able to
initiate normally. The presence of more replicators
than needed to complete DNA replication is a form
of redundancy to ensure the complete replication of
each chromosome.

The steps in the

formation of the prereplicative
complex (pre-RC).

The assembly of the pre-RC is an

ordered process that is initiated by the
association of the origin recosnition
complex
with the replicator. Once
bound to the replicator, ORC recruits
at least two additional proteins, Cdc6
and Cdtl. These three proteins function
together to recruit the putative
eukariotic DNA helicase-the Mcm2-7
complex to complete the formation of
the pre-RC.

Activation of the pre-RC

leads to the assembly of the
eukaryotic replication fork

As cells enter into the S phase of the cell

cycle, Cdk and Ddk phosphorylate
replication proteins to trigger the
initiation of replication. The events that
lead to DNA unwinding at the origin are
poorly understood but are likely to
require the activity of the Mcm complex
and result in the recruitment of a number
of auxiliary replication factors and DNA
Pols. DNA Pol is only recruited after
DNA Pol and Pol and once present at
the origin, DNA Pol synthesizes an
RNA primer and bdefly extends it.

Activation of the pre-RC

leads to the assembly of the eukaryotic replication fork

The resulting primer: template junction is

recognized by the eukaryotic sliding
clamp loader (RF-C), which assembles a
sliding clamp (PCNA) at these sites.
DNA Pol 6 recognizes this primer and
begins leading strand synthesis. After a
period of DNA unwinding, DNA Pol
edodmase synthesizes additional primers,
which allow the initiation of lagging
strand DNA synthesis by either DNA Pol 
or others polimerase.
Here we illustrate Pol 6 on the leading strand and Pol 
on the lagging strand.

Activitatea de corectare (editare)

Replicarea ADN necesit cooperarea mai multor proteine,
care constituie o main de replicare multienzimatic la nivelul
furcii de replicare, cataliznd sinteza ADN ; ADN polimeraza
realizeaz replicarea unei matrie ADN cu o remarcabil fidelitate,
nregistrnd mai puin de o eroare pentru fiecare 107 baze parcurse
(citite). Acest lucru este posibil deoarece enzima elimin propriile
sale erori de polimerizare deplasndu-se dea lungul ADN (etap de
proofreading sau de corectare a greelilor);
Activitatea de corectare pe care o realizeaz ADN polimeraza
face ca enzima s fie incapabil s realizeze sinteza de novo a unuei
noi catene ADN. Sinteza ADN este amorsat de ctre o ARN
polimeraz, numit primaz, care sintetizeaz fragmente scurte de
ARN numite amorce sau primeri care sunt apoi (la sfritul
procesului) nlturate i nlocuite cu ADN;

Repararea ADN prin mecanismul de editare

Rarele erori de copiere care scap mainriei de
replicare a ADN sunt luate n primire de ctre sistemele
proteice de reparare a nemperecherilor, care controleaz
secvenele de ADN nou sintetizate i repar erorile de copiere.
Precizia global a replicrii ADN, innd cont de repararea
nemperecherilor, este de o eroare pentru 109 nucleotide
copiate;

Activitatea de proofreading a ADN

polimerazei corecteaz erorile de copiere

Modelul schematic al activitii de

proofreading a ADN polimerazei

Telomeraza previne scurtarea catenelor

ntrziate n timpul replicrii eucariotelor

Structura ADN in
telomeri

Rolul topoizomerazelor n replicarea ADN

Moleculele de ADN se pot ndoi i ncolci n spaiu
conducnd la modificri de topologie cum ar fi formarea
supra-rsucirilor
Topoizomerazele sunt enzime care controleaz
topologia ADN i realizeaz etape eseniale n diferite etape
ale replicrii avnd drept rezultat nlturarea
constrngerilor.

Topoizomeraza de tip I relaxeaz ADN prin

inducerea de tieturi la nivelul unei singure
catene a duplexului ADN

Separarea topoizomerilor ADN SV40 DNA

evideniaz existena mai multor topoizomeri
cu un numr diferit de suprarsuciri

Topoizomeraza de tip II modific topologia ADN

prin scindarea si resudarea ambelor catene

Modelul activitii catalitice a topoizomerazei II

(ADN giraza) din E. coli

ADN giraza nltur super-rsuricirile pozitive

care apar n mod normal la nivelul furcii de
replicare

Separarea moleculelor de de ADN circular

replicate este realizat de topoizomeraza de tip II

Topoizomeraza de tip II separ

cromatidele lineare surori

Impachetarea ADN: histonele chaperone si caile de

asamblare nucleosomala

 la EK, impachetarea DNA in cromatina este esentiala

pentru viabilitatea celulara; cantitatea de ADN dintr-o
celula EK este de ~2 m
materialul genetic este compactat cu ajutorul
unor proteine bazice = histone, pentru a incapea
in spatiul nuclear stramt
histonele - 5 clase: H1, H2A, H2B, H3 si H4
studii de difractie cu raze X
cromatina(cromozomii interfazici) prezinta structuri repetabile la intervale de 10nm
fiecare unitate repetitiva = nucleosom, cuprinde un octamer histonic = 2(H2A,H2B, H3,
H4)
modalitatea de biosinteza a ADN este replicarea, are loc in faza S- astfel, suvita ADN
este desfacuta de helicaza, iar sinteza de catre polimeraza necesita dezmembrarea
tranzitorie a contactelor histona-AND si transferul histonelor parentale in urma furcii
de replicare
FACT = este un complex proteic nuclear,
conservat = factor necesar elongarii transcriptiei pe
matritele cromatinei; destabilizeaza interactia dintre
dimerii H2A/H2B si H3/H4 a nucleosomuluireorganizarea
Structurii nucleosomului
FACT face parte din complexul de progresie a furcii de
replicare

 FACT faciliteaza transcriptia cromatinei

are 2 subunitati conservate in evolutie
la toate EK: - Spt16 ; - SSRP1
la S. cerevisiae proteina
Pob3 este omoloaga SSRP1
mutatiile hipomorfice de la drojdii
-SPT16 si POB3, cat si mutatiile ce slabesc
interactia FACT cu DNA polimerazasusceptibilitatea la HU(hidroxiuree)
-inhibitor al replicarii DNA,prin depletia dNTP
FACT leaga direct complexul primaza-DNA polimeraza, care creeaza primeri pentru
initierea replicarii catenei leading a ADN si sinteza fragmentelor Okazaki
FACT dislocuie unul din cei 2 dimeri H2A-H2B, care paraseste tetramerul (H2AH2B)2, acesta fiind un obstacol ce trebuie depasit de catre polimerazaII
ASF1 = anti-silencing function 1- histona chaperone implicata in replicarea
dependenta/independenta de asamblarea nucleosomala
ASF1 este asociat stabil cu MCM(minicromosome maintenance) si cu histonele H3-H4
multe histone purificate din acest complex sunt histone parentale, continand PTMmodificari post-translationale= trimetilare H3K9 si acetilare H4K16

 absenta ASF1 in celulele eucariote acumulare a celulelor in faza S,

generarea cromatinei imature sensibila la nucleaza, o descrestere in
incorporarea de Brd-U(bromodeoxyuridina) in DNA nascent si leziuni
spontane in DNA aberatii in segregarea cromozomilor si letalitate
celulara.
acest fenotip apare cand CAF-1=factorul de asamblare al cromatinei, este
inactivat
CAF-1 are 3 subunitati conservate (p150, p60 si RbAp48) implicate in
depozitarea noilor histone H3-H4 sintetizate, in urma furcii de replicare,
prin interactia cu PCNA= antigenul nuclear al celulelor proliferative
PCNA (Proliferating Cell Nuclear Antigen) este o proteina homo-dimera ce
inconjoara ANDdc, si este un factor de pocesivitate pentru DNA
polimeraze, poate servi drept platforma mobila pentru depozitarea de
novo a histonelor

Duplicarea cromatinei implica 2 reactii concertate care au loc rapid in timpul

trecerii furcii de replicare a ADN:
- transferul histonelor parentale sau a histonelor pre-existente (blue-disk) in
urma furcii de replicare, care are loc in general in duplexurile generate prin
sinteza oricarei catene leading sau lagging;
- golurile din nucleosomi, sunt umplute prin depozitarea noilor histone
sintetizate(orange-disks) care sunt acetilate la cateva reziduuri de lizina.

De-a lungul elongarii transcriptiei, histonele parentale sunt transferate in

duplexul DNA in urma RNA polimerazei II. Golurile ocazionale din
nucleosomi care apar in genele inalt transcriptibile sunt refacute prin
depozitarea variantei histonice H3.3 independenta de replicare, impreuna
cu histona H4.

 HAT=histon-acetiltransferaza tip B, enzima care acetileaza histonele in special la

incorporarea lor in cromatina; reziduurile acetilate apartin domeniilor NH2terminale ale histonelor H3 si H4
absenta HAT nu e letala, dar confera sensibilitate la agenti genotoxici
HAT este asociat cu Asf1, H3-H4
recent s-au identificat noi situsuri de acetilare :
* Lys91 din H4 (H4K91)in vitro slabeste legarea dimerilor H2A-H2B la
tetramerii (H3-H4)2;
*Lys56 din H3(H3K56) histonele depozitate in urma furcii de replicare, de-a
lungul fazei S, sunt K56 H3 acetilate de catre o HAT tip B=Rtt109
la S. cerevisiae toate histonele H3 nou depozitate sunt K56, si deacetilate global in
stadiile tarzii ale ciclului celular
aceasta acetilare/deacetilare stabilitate genomica
abilitatea celulelor de drojdii de a supravietui la
agenti genotoxici in timpul replicarii
punctele de verificare(checkpoint) degradeaza deacetilazele H3K56( Hst3 si Hst4),
pastreaza acetilarea H3K56 si promoveaza supravietuirea celulara

 variantele H3.1/H3.2 sunt predominant asociate cu CAF-1, in timp ce H3.3

este in special legata la HIRA( in celulele proliferative)
H3.3 este predominant incorporata in genele care sunt transrise
activ(sintetizata pe durata intregului ciclu celular)
Senescenta celulara, in celulele umane, este asociata cu o condensare globala
a cromatinei, unde fiecare cromozom este impachetat intr-o structura
foarte compacta cunoscuta drept SAHF = foci heterocromatici asociati
senescentei
SAHF impune oprirea ciclului celular
proteinele chaperone ASF1 si HIRA depoziteaza histonele H3-H4 in
DNAcondensare nucleosomala
formarea SAHF
CONCLUZII:
*sinteza DNA asociata cailor de asamblare este esentiala viabilitatii celulare
contribuind la repararea leziunilor DNA;
*acetilarea histonelor mentine stabilitatea genomica si rezistenta la agenti
genotoxici ;

Leziunile ADN, repararea lor i rolul

acestora n carcinogenez
O secven de ADN poate fi schimbat n timpul copierii
datorit erorilor introduse de ctre ADN polimeraz n timpul
replicrii i de ctre agenii de mediu: ageni mutageni chimici
i radiaiile;
Necorectarea lor face ca aceste modificri s interfere cu
capacitatea funcional a celulei;
Leziunile ADN pot fi reparate prin mai multe
mecanisme;
Toi carcinogenii determin modificri ale secvenei
ADN i astfel lezarea i repararea sunt aspecte importante n
apariia diferitelor forme de cancer;
Sistemele de reparare a ADN de la procariote i
eucariote sunt analoge.

Tipuri generale de leziuni din structura ADN i

cauzele acestora

Repararea leziunilor ADN (1)

Erorile de replicare a ADN i reaciile chimice care modific
nucleotidele n ADN antreneaz modificri n secvena nucleotidic a
ADN. Dac aceste modificri nu sunt corectate eficient, ele dau
natere la mutaii, marea majoritate a acestora fiind nefaste pentru
organism. Informaia genetic poate fi stabil stocat n secvena
ADN numai n cazul n care diferitele enzime de reparare analizeaz
n permanen secventa ADN i corecteaz erorile de replicare
nlocuind nucleotidele modificate. ADN poate uor reparat deoarece
o caten poate fi reparat utiliznd informaia de pe catena
complementar;

Repararea leziunilor ADN (2)

Leziunile ADN datorate reaciilor chimice i
radiaiilor UV sunt corectate de ctre diferite enzime,
care recunosc ADN modificat i excizeaz un fragment
scurt din catena de ADN care l conine. Secvena de ADN
nlturat este resintetizat de ctre o ADN polimeraz
de reparare, care folosete drept matri catena fr
leziuni. ADN ligaza sudeaz fragmentele de ADN pentru
a completa procesul de reparare.

Carcinogeni chimici care acioneaz asupra ADN

direct sau dup activare

Efectul carcinogen al compuilor chimici este corelat cu

capacitatea mutagen

Dezaminarea bazelor conduce la formarea spontan a

mutaiilor punctiforme

Repararea mperecherilor nepotrivite la nivelul unui

defect punctiform

Repair of Mismatched Bases

Even with editing by the polymerase, an occasional mismatch in the DNA will occur. In this
case, the error is repaired by a system called the mutHLS repair pathway. This is
diagrammed in the figure below:

Baze modificate
chimic: dimeri timintimin

Radiaiile UV pot induce

formarea unor legturi covalente
ntre resturile de timin. Dimerii
de
timin
rezultai,
ciclobutiltimin, pot fi corectate
prin mecanismul de exciziereparare.

Repair of Pyrimidine Dimers in DNA

Formation of Pyrimidine Dimers
Irradiation of DNA with ultraviolet light
can cause the formation of dimers that
are
covalently
linked
adjoining
pyrimidines. The most common are
thymine dimers, although TC and CC
dimers can occur. Pyrimidine dimers are
found in two forms:
In both cases, the presence of the dimers
in the DNA is damaging. It may cause a
mispairing as the strand is being copied
or may stop replication
altogether. The most common result is
the first and mutations then arise.
Therefore, systems have evolved to
repair this damage. Because we live on a
planet bathed in a significant amount of
UV light, it is not surprising that more
than one repair system can deal with this
damage.

Mecanismul UvrABC
de excizie-repair a
ADN la E. coli

Excision Repair
Thymine dimers can be removed from DNA by excising them (cutting them out),
using a complex of proteins called the uvrABC system. The sequence of events is
shown in the figure below:

ndeprtarea Uracil din ADN

De ce uracilul n ARN i timina in

ADN?"
Exista diferite cai prin care uracilul
poate sa apara n ADN.

Modaliti de ndeprtarea
a Uracilului

1. U este nlturat de ctre uracil-DNA Nglycosidase care excizeaz U prin tierea

legturilor dintre baz i restul glucidic;
2. ndeprtarea U induce apariia unui situs
apirimidinic. Acesta este recunoscut de ctre
enzima apyrimidinic (AP) endonuclease, care
taie catena ADN determinnd apariia unei bree
monocatenare;
3. ADN polimerase I (pol I) interacioneaz cu
aceast bre folosind activitatea 5-3'
exonucleazic i 5- 3' polimerazic pentru a
traduce deplasarea nick.Aceasta se realizeaz
prin degradare i sintez simultan. Rezultatulrepararea regiunii afectate;
4. DNA ligase sudeaz capetele breei i aduce
ADN n starea sa iniial.

Photoreactivation
Pyrimidine dimers can be
eliminated by photoreactivation,
using the energy of light to split
the dimer apart. The enzyme
reaction is diagrammed in this
figure:
The enzyme is called
photolyase and uses visible light
(l = 370 nm) to oxidize the
cyclobutane ring. The light
energy is harvested by a
chromophore and that energy is
used to produce a free radical
(FADH) that carries out the
oxidation, returning the DNA to
its normal state. Examples of
these enzymes are found in both
prokaryotic and eukaryotic
systems.

Repararea capetelor
ADN neomolog

Eucariotele au sisteme de reparare a ADN

analoge cu cele de la E. coli
Proteinele Mut i MutS i MutL umane - omologi ai proteinelor
bacteriane se leag att la bazele nemperechiate ct i la micile inserii sau deleii.
Clivarea n 3 fie n 5 fa de nemperechere; o exonucleaz ndeprteaz 100 la
200 nucleotide. ADN polimeraza i ADN ligaz sunt principalele responsabile
pentru repararea catenei
Mecanisme similare de excizie-reparaie sunt folosite de diferite
organisme. Strategia de baz este determinarea de mutani cu sensibilitate
crescut la UV i ali ageni care determin leziuni ADN. La S. Cerevisiae - genele
radiosensibile (RAD). Tehnici de inginerie genetic - generai mutani de linii
celulare CHO cu sensibilitate anormal la UV sau la mitomicina C. Mutaiile n 8
grupuri de complementare (8 gene diferite implicate excizie-reparare la hamster.
Evideniere experimental de gene umane omologe cu genele RAD3 i
RAD10 de la drojdii. Proteinele umane omologe cu RAD10 conin i o regiune
similar cu o parte a UvrC de la E. coli.
Cuplarea transcripiei cu repararea evideniat pregnant n cazul genelor
intens transcrise

Sistemele inductibile de reparare a ADN

predispoziie spre erori
Numeroase alterri ale ADN - sistemele de reparare sunt saturate
activare de sisteme de repare inductibile.
Sistemul de reparare SOS de la bacterii; din pcate acest furnizeaz i el
predispouiie la erori (error-prone).
Sistem folosit numai n ultim instan.
Celulele animale posed sisteme de reparare inductibile
Principalul mecanism de reparare a fragmentelor dublu catenare rupte
determin inducerea de erori.
Se consider c multe dintre mutaiile care apar i se pstreaz n
structura ADN sunt mai degrab consecina alterrilor indirecte dect directe
asupra ADN.
Celula posed mecanisme care vor sesiza alterrile i vor stopa replicarea
Punct principal de control reprezentat de proteina p53, care acioneaz pentru a
stopa ciclul celular dac ADN este alterat

Post-Replication Repair (SOS Repair)

Recombinarea secvenelor de ADN omologe

Recombinarea ofer o modalitate prin care genomul poate

schimba i genera noi combinaii de gene
Recombinarea omolog permite schimbarea de blocuri de
gene ntre cromozomii omologi, acesta fiind un mecanism de
generare a diversitii genetice.
Recombinarea apare la ntmplare ntre dou secvene
omologe i frecvena de recombinare ntre dou situri este
proporional cu distana dintre aceste situri.

Prezentare (1)
n timpul recombinrii omologe, dou molecule ADN duplex sunt
scindate i catenele sunt schimbate. Acest proces, care apare aleator pe toat
lungimea genoamelor tuturor organismelor, joac un rol important n generarea
diversitii genetice;
Ruperea dublu-catenelor iniiaz n majoritatea cazurilor recombinare
omolog. Ruptura devine se lrgete devenind bre formnd capete 3 de
recombinare care invadeaz alte duplexuri. Sinteza reparatorie a regiunilor lips
formeaz un intermediar care conine dou jonciuni Holliday ncruciate.
Rezolvarea acestui intermediar apare prin rotaie urmat de clivarea i ligarea
celor dou catene ale fiecrei jonciuni Holliday.
La E. coli captul de recombinare creat de complexul enzimatic RecBCD
este stabilizat prin legarea proteinei RecA. Catalizat de RecA, captul
monocatenar 3 se mperecheaz apoi i invadeaz un segment de duplex omolog,
formnd un intermediar coninnd dou regiuni de heteroduplex ADN i dou
jonciuni Holliday. Dup migrarea ramurii, catalizat de ctre RuvA i RuvB,
dou catene ale fiecrei jonciuni sunt tiate de ctre endonucleaz RuvC i apoi
ligate pentru a forma dou molecule ADN duplex

Prezentare (2)

Deoarece celulele eucariote exprim proteine omologe celor de

recombinare de la E. coli, se consider c toate celulele realizeaz
procesul de recombinare printr-un mecanism molecular similar;
Recombinazele situs-specifice recunosc, taie i recombin
secvene de ADN omologe scurte la dou tipuri diferite de molecule de
ADN;
n timpul recombinrii situs-specifice catalizat de ctre proteina
Cre a fagului P1, se formeaz legturi fosfotirozin ntre moleculele Cre
i capetele 3-hidroxil ale duplexurilor ADN. Integrarea fagului ,
catalizat de ctre integraz , se consider c apare printr-un similar.

Structura inter-catenar Holliday reprezint

un intermediar de recombinare (partea I)

Structura inter-catenar Holliday reprezint

un intermediar de recombinare (partea II)

Recombinarea plasmidelor ADN

Scindare dublu catenar n iniierea

recombinrii ADN (partea I)

Scindare dublu catenar n iniierea

recombinrii ADN (partea II)

Iniierea recombinrii de ctre enzima RecBCD

Proteina RecA coordoneaz modificari catenare,

mperechere omolog i formarea unei structuri
tip Holliday

Migrarea catenei i rezolvarea structurilor

Holliday depinde de proteinele Ruv

Aciunea proteinelor E. coli asupra migrii

catenelor i rezolvrii structurilor

Proteina Cre i alte recombinaze catalizeaz

recombinarea situs specific

Mecanismul recombinrii situs specifice

realizat de Cre-loxP

Recombinarea situs specific-modelul

intermediarului II Cre-DNA