Bien avant que la structure de l’ADN ne soit connue, les scientifiques se sont interrogés sur la capacité des organismes à créer des copies fidèles d’eux mêmes et, plus tard, sur la capacité des cellules à produire plusieurs copies identiques de macromolécules complexes. La spéculation à propos de ces problèmes était centrée sur le concept d’un « modèle », une structure qui permettrait aux molécules d’être alignées dans un ordre spécifique et jointes, pour créer une macromolécule avec une séquence et une fonction uniques. Les années 1940 ont apporté la révélation que l’ADN était ce modèle (la molécule génétique), mais ce n’est que lorsque Watson et Crick ont décrit sa structure, que la façon dont l’ADN pourrait servir de modèle pour la réplication et la transmission de l’information génétique est devenue claire.: chaque brin est le complément de l’autre. Les règles d’appariement de bases strictes signifient que chaque brin fournit le modèle pour un brin frère avec une séquence prévisible et complémentaire

Les nucléotides (les éléments constitutifs des acides nucléiques) et les propriétés fondamentales du processus de réplication de l’ADN et les mécanismes utilisés par les enzymes qui le catalysent se sont révélés essentiellement identiques chez toutes les espèces. La recherche précoce sur la réplication de l’ADN bactérienne et ses Enzymes ont permis d’établir les propriétés fondamentales qui sont applicables à la réplication de l’ADN chez tous les organismes.

La réplication de l’ADN est semiconservative Chaque brin d’ADN sert de modèle pour la synthèse d’un nouveau brin, produisant deux nouvelles molécules d’ADN, chacune avec un nouveau brin et un ancien brin. C’est une réplication semi-conservative. Watson et Crick ont proposé l’hypothèse d’une réplication semi-conservatrice peu après la publication de leur article de 1953 sur la structure de l’ADN, et leur l’hypothèse a été prouvé par des expériences conçues par Matthew Meselson et Franklin Stahl en 1957.

Meselson et Stahl ont cultivé des cellules d’E. coli pendant de nombreuses générations dans un milieu où la seule source d’azote (NH4Cl) contenait 15N, l’isotope «lourd» de l’azote. , au lieu de l’isotope « léger » normal de 14N, plus abondant dans la nature. L’ADN isolé à partir de ces cellules avait une densité supérieure d’environ 1% à celle de l’ADN [14N] normal (figure 25-2a). Bien que ce soit seulement une petite différence, un mélange d’ADN lourd [15N] et d’ADN léger [14N] peut être séparé par centrifugation jusqu’à l’équilibre dans un  gradient de densité de  chlçorure de césium. Les cellules d’ E. coli cultivées dans le milieu 15N étaient transférés dans un milieu frais contenant uniquement l’isotope 14N, où ils ont été laissés pousser jusqu’à ce que la population cellulaire ait juste doublé. L’ADN isolé de ces cellules de première génération ont formé une seule bande dans le gradient de densité de CsCl gradient à une position indiquant que le double helice des molécules d’ADN des cellules filles étaient des hybrides contenant un nouveau brin 14N et un parent 15N.

Ce résultat plaide contre la réplication conservatrice, une hypothèse alternative dans laquelle la molécule d’ADN fille consisterait en deux brins d’ADN nouvellement synthétisés et l’autre contiendrait les deux brins parentaux, cela ne donnerait pas de molécules d’ADN hybrides dans l’expérience de Meselson-Stahl. L’hypothèse de réplication semiconservative a été soutenue dans la prochaine étape de l’expérience. Les cellules ont à nouveau été autorisées à doubler en nombre dans le milieu 14N. Le produit d’ADN isolé de ce second cycle de réplication exhibait deux bandes dans le gradient de densité, l’une avec une densité égale à celle de l’ADN léger et l’autre avec la densité de l’ADN hybride observée après le doublement de la première cellule.

La réplication commence à une origine et se poursuit de manière bidirectionnelle  :

Suite à la confirmation du mécanisme semi-conservateur de la réplication, une foule de questions ont surgi. Les brins d’ADN parents sont-ils complètement déroulés avant que chacun soit répliqué? La réplication commence-t-elle au hasard à un endroit ou à un point unique? Après l’initiation à un point donné
la réplication se poursuit elle dans une direction ou dans deux directions? Une indication précoce que la réplication est un processus hautement coordonnée dans lequel les brins parents sont simultanément déroulés et répliqués a été fourni par John Cairns .Il a créé des E. coli avec un ADN radioactif par culture de cellules dans un milieu contenant la thymidine marquée avec du tritium (3H). Quand l’ADN a été soigneusement isolé, étalé et recouvert d’une émulsion  photographique pendant plusieurs semaines, les résidus de thymidine ont généré des «traces» de grains d’argent, produisant une image de la molécule d’ADN. Ces traces ont révélé que le chromosome intact de E. coli est un grand cercle de 1,7 mm de long. L’ADN radioactif isolé des cellules durant la réplication a montré un boucle supplémentaire (figure 25-3a). Cairns a conclu que la boucle résulte de la formation de deux copies radioactives des brins, chacun complémentaire d’un brin parent. Un ou les deux extrémités de la boucle sont des points dynamiques, appelés fourches de réplication, où l’ADN parent est en train d’être déroulé et les brins séparés rapidement reproduits.

Les résultats de Cairns ont démontré que les deux brins d’ADN sont répliqué simultanément, et une variation sur son expérience a indiqué que la réplication des chromosomes des batéries est bidirectionnelle: les deux extrémités de la boucle ont des fourches de réplication actives. Pour savoir si les boucles de réplication débutaient à un point unique dans les repères d’ADN a requis la connaissance de ces repères le long de la molécule d’ADN. Ceux-ci ont été fournis par une technique appelée cartographie de dénaturation, développé par Ross Inman et ses collègues. Utilisant le 48,502 pb chromosomes  du bactériophage λ, Inman a montré que L’ADN pourrait être sélectivement dénaturé à des séquences exceptionnellement riche en paires de bases A = T, générant un  modèle de bulles à simple brin reproductible :

ADN isolé contenant des boucles de réplication peut être partiellement dénaturé de la même manière. Cela permet la mesure et la cartographie des différentes position et la progression des fourches de réplication en utilisant les régions dénaturées comme des points de référence. La technique a révélé que dans ce système les boucles de réplication démarrent toujours à un point unique, qui a été appelé  l’origine. Il a également confirmé la précédente observation que la réplication est généralement bidirectionnelle. Pour les molécules d’ADN circulaires, les deux fourches de réplication se rencontrent à un point du côté du cercle opposé à l’origine. Les origines spécifiques de la réplication ont depuis été identifiées et caractérisées dans les bactéries et eucaryotes inférieures.
La synthèse d’ADN a lieu dans une direction 5 ‘→ 3′ et est semi-continue. Un nouveau brin d’ADN est toujours synthétisé dans le sens  5′ →3′, avec le 3′ OH libre comme le point à laquelle l’ADN est allongé. Parce que les deux brins d’ADN sont antiparallèles, le brin servant de modèle est lu à partir de son extrémité 3′ vers son extrémité 5′.
Si la synthèse se poursuit toujours dans le sens 5′→ 3′, comment les deux brins peuvent-ils être synthétisés simultanément? Si les deux brins sont synthétisés en continu pendant que la fourche de réplication se déplace, alors un brin devrait subir la synthèse de 3′→5′. Ce problème a été résolu par Reiji Okazaki et ses collègues dans les années 1960. Okazaki a trouvé que l’un des nouveaux brins d’ADN est synthétisé en petits  morceaux séparés, maintenant appelés fragments d’Okazaki. Ce travail finalement conduit à la conclusion qu’un brin est synthétisé en continu et l’autre en discontinu.

Le brin continu, ou brin principal, est celui dans lequel la synthèse se déroule dans le sens 5′→3′ c.à.d dans la même direction que le mouvement de la fourche de réplication. Le brin discontinu, ou brin secondaire (lagging), est celui dans lequel  la synthèse se déroule de 5′ →3′ mais dans la direction opposée à celle du mouvement de la fourche. Les fragments d’Okazaki ont une longueur de quelques centaines à quelques milliers de nucléotides, en fonction du type de cellule. La synthèses des brins leaders et secondaires est étroitement coordonné.

L’ADN est dégradé par les nucléases:

Pour expliquer l’enzymologie de la réplication de l’ADN, nous introduisons d’abord les enzymes qui dégradent l’ADN plutôt que de le synthétisent. Ces enzymes sont connues sous le nom de nucléases, ou DNases si elles sont spécifiques de l’ADN plutôt que de l’ARN. Chaque cellule contient plusieurs nucléases différentes, appartenant à deux grandes classes: les exonucléases et les endonucléases. Les exonucléases dégradent les acides nucléiques d’une
extrémité de la molécule. Beaucoup fonctionnent seulement dans le sens 5′ →3′ ou le sense opposé 3′ →5′, en enlevant des nucléotides seulement à partir de l’extrémité 5′ ou 3′, respectivement, d’un brin d’ADN double brin ou d’un ADN simple brin. Les endonucléases peuvent commencer à dégrader à des sites internes spécifiques dans un brin ou une molécule d’ADN, en le réduisant en fragments de plus en plus petits. Quelques exonucléases et endonucléases dégradent seulement l’ADN simple brin. Il existe quelques classes importantes d’endonucléases qui ne coupent qu’à des séquences nucléotidiques spécifiques (telles que les endonucléases de restriction qui sont si importantes en biotechnologie. Vous rencontrerez de nombreux types de nucléases dans ce chapitre et les suivants.

L’ADN est synthétisé par des ADN polymérases :

La recherche d’une enzyme capable de synthétiser l’ADN a commencé en 1955. Les travaux d’Arthur Kornberg et de ses collègues ont condui à la purification et à la caractérisation de l’ADN polymérase provenant des cellules d’ E. coli, une enzyme à polypeptide unique appelée maintenant ADN polymérase I. Beaucoup plus tard, les chercheurs ont découvert qu’E. coli contenaient au moins quatre autres ADN polymérases distinctes, décrites ci-dessous. Des études détaillées de l’ADN polymérase I ont révélé des caractéristiques du processus de synthèse de l’ADN qui sont maintenant connues pour être communes à toutes les ADN polymérases. La réaction fondamentale est un transfert de groupe phosphoryle :

dNMP et dNTP sont désoxynucleoside 5′-monophosphate et 5′-triphosphate, respectivement. Le nucléophile est le groupe 3′-hydroxyle du nucléotide au niveau de l’extrémité 3′ du brin croissant. L’attaque nucléophile se produit au niveau du phosphore α du désoxynucléoside 5′-triphosphate entrant. Le pyrophosphate inorganique est libéré dans la réaction.

La réaction semble se dérouler avec seulement un changement minime de l’énergie libre, étant donné qu’une liaison phosphodiester est formée aux dépens d’un anhydride phosphate un peu moins stable. Cependant, les interactions non-covalentes d’empilement de bases et d’appariements de bases fournissent une stabilisation supplémentaire au produit d’ADN allongé par rapport au nucléotide libre. En outre, la formation de produits est facilitée dans la cellule par les 19 kJ / mol générés lors de l’hydrolyse subséquente du produit pyrophosphate par l’enzyme pyrophosphatase.

Early work on DNA polymerase I led to the definition of two central requirements for DNA polymerization. First, all DNA polymerases require a template. The polymerization reaction is guided by a template DNA strand according to the base-pairing rules predicted by Watson and Crick: where a guanine is present in the template, a cytosine deoxynucleotide is added to the new strand, and so on. This was a particularly important discovery, not only because it provided a chemical basis for accurate semiconservative DNA replication but also because it represented the first example of the use of a template to guide a biosynthetic reaction. Second, the polymerases require a primer. A primer is a strand segment (complementary to the template) with a free 3
-hydroxyl group to which a nucleotide can be added; the free 3
end of the primer is called the primer terminus. In other words, part of the new strand must already be in place: all DNA polymerases can only add nucleotides to a preexisting strand. Most primers are oligonucleotides of RNA rather than DNA, and specialized enzymes synthesize primers when and where they are required. After adding a nucleotide to a growing DNA strand, a DNA polymerase either dissociates or moves along the template and adds another nucleotide. Dissociation and reassociation of the polymerase can limit the overall polymerization rate—the process is generally faster when a polymerase adds more nucleotides without dissociating from the template. The average number of nucleotides added before a polymerase dissociates defines its processivity. DNA polymerases vary greatly in processivity; some add just a few nucleotides before dissociating, others add many thousands. Nucleotide Polymerization by DNA Polymerase Replication Is Very Accurate Replication proceeds with an extraordinary degree of fidelity. In E. coli, a mistake is made only once for every 109 to 1010 nucleotides added. For the E. coli chromosome
of ~4.6  106 bp, this means that an error occurs only once per 1,000 to 10,000 replications. During polymerization, discrimination between correct and incorrect nucleotides relies not just on the hydrogen bonds that specify the correct pairing between complementary bases but also on the common geometry of the standard AUT and GmC base pairs (Fig. 25–6). The active site of DNA polymerase I accommodates only base pairs with this geometry. An incorrect nucleotide may be able to hydrogen-bond with a base in the template, but it generally will not fit into the active site. Incorrect bases can be rejected before the phosphodiester bond is formed. The accuracy of the polymerization reaction itself, however, is insufficient to account for the high degree of fidelity in replication. Careful measurements in vitro have shown that DNA polymerases insert one incorrect nucleotide for every 104 to 105 correct ones. These mistakes sometimes occur because a base is briefly in an unusual tautomeric form (see Fig. 8–9), allowing it to hydrogen-bond with an incorrect partner. In vivo, the error rate is reduced by additional enzymatic mechanisms. One mechanism intrinsic to virtually all DNA polymerases is a separate 3
n5
exonuclease activity that double-checks each nucleotide after it is added. This nuclease activity permits the enzyme to remove a newly added nucleotide and is highly specific for mismatched base pairs (Fig. 25–7). If the polymerase has added the wrong nucleotide, translocation of the enzyme to the position where the next nucleotide is to be added is inhibited. This kinetic pause provides the opportunity for a correction. The 3
n5
exonuclease activity removes the mispaired nucleotide, and the polymerase begins again. This activity, known as proofreading, is not simply the reverse of the polymerization reaction (Eqn 25–1), because pyrophosphate is not involved. The polymerizing and proofreading activities of a DNA polymerase can be measured separately. Proofreading improves the inherent accuracy of the polymerization reaction 102- to 103-fold. In the monomeric DNA polymerase I, the polymerizing and proofreading activities have separate active sites within the same polypeptide. When base selection and proofreading are combined, DNA polymerase leaves behind one net error for every 106 to 108 bases added. Yet the measured accuracy of replication in E. coli is higher still. The additional accuracy is provided by a separate enzyme system that repairs the mismatched base pairs remaining after replication. We describe this mismatch repair,
along with other DNA repair processes, in Section 25.2.
E. coli Has at Least Five DNA Polymerases
More than 90% of the DNA polymerase activity observed
in E. coli extracts can be accounted for by DNA polymerase
I. Soon after the isolation of this enzyme in 1955,
however, evidence began to accumulate that it is not
suited for replication of the large E. coli chromosome.
First, the rate at which it adds nucleotides (600 nucleotides/
min) is too slow (by a factor of 100 or more)
to account for the rates at which the replication fork
moves in the bacterial cell. Second, DNA polymerase I
has a relatively low processivity. Third, genetic studies
have demonstrated that many genes, and therefore
many proteins, are involved in replication: DNA polymerase
I clearly does not act alone. Fourth, and most
important, in 1969 John Cairns isolated a bacterial strain
with an altered gene for DNA polymerase I that produced
an inactive enzyme. Although this strain was abnormally
sensitive to agents that damaged DNA, it was
nevertheless viable!
A search for other DNA polymerases led to the
discovery of E. coli DNA polymerase II and DNA
polymerase III in the early 1970s. DNA polymerase II
is an enzyme involved in one type of DNA repair (Section
25.3). DNA polymerase III is the principal replication
enzyme in E. coli. The properties of these three
DNA polymerases are compared in Table 25–1. DNA
25.1 DNA Replication 955
DNA polymerase I
OH
Before the polymerase
moves on, the cytosine
undergoes a tautomeric
shift from C* to C. The
new nucleotide is now
mispaired.
is a rare tautomeric
form of cytosine (C*)
that pairs with A and
is incorporated into
the growing strand.
The mispaired 3
-OH
end of the growing
strand blocks further
elongation. DNA
polymerase slides back
to position the
mispaired base in the
3
→5
exonuclease
active site.
The mispaired
nucleotide is removed.
DNA polymerase slides
forward and resumes its
polymerization activity.
DNA polymerase
active site
3
→5
(proofreading)
exonuclease

FIGURE 25–7 An example of error correction by the 3
n5
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
n5
exonuclease activity, then resumes its polymerase activity
in the 5
n3
direction.
polymerases IV and V, identified in 1999, are involved
in an unusual form of DNA repair (Section 25.2).
DNA polymerase I, then, is not the primary enzyme
of replication; instead it performs a host of clean-up
functions during replication, recombination, and repair.
The polymerase’s special functions are enhanced by its
5
n3
exonuclease activity. This activity, distinct from
the 3
n5
proofreading exonuclease (Fig. 25–7), is located
in a structural domain that can be separated from
the enzyme by mild protease treatment. When the
5
n3
exonuclease domain is removed, the remaining
fragment (Mr 68,000), the large fragment or Klenow
fragment (Fig. 25–8), retains the polymerization and
proofreading activities. The 5
n3
exonuclease activity
of intact DNA polymerase I can replace a segment of
DNA (or RNA) paired to the template strand, in a
process known as nick translation (Fig. 25–9). Most
other DNA polymerases lack a 5
n3
exonuclease
activity.
DNA polymerase III is much more complex than
DNA polymerase I, having ten types of subunits (Table
25–2). Its polymerization and proofreading activities reside
in its  and  (epsilon) subunits, respectively. The

subunit associates with  and  to form a core polymerase,
which can polymerize DNA but with limited
processivity. Two core polymerases can be linked by
956 Chapter 25 DNA Metabolism
TABLE 25–1 Comparison of DNA Polymerases of E. coli
DNA polymerase
I II III
Structural gene* polA polB polC (dnaE)
Subunits (number of different types) 1 7 10
Mr 103,000 88,000† 791,500
3
n5
Exonuclease (proofreading) Yes Yes Yes
5
n3
Exonuclease Yes No No
Polymerization rate (nucleotides/s) 16–20 40 250–1,000
Processivity (nucleotides added 3–200 1,500 500,000
before polymerase dissociates)
*For enzymes with more than one subunit, the gene listed here encodes the subunit with polymerization activity. Note that dnaE
is an earlier designation for the gene now referred to as polC.
†Polymerization subunit only. DNA polymerase II shares several subunits with DNA polymerase III, including the , , , , ,
and
subunits (see Table 25–2).
TABLE 25–2 Subunits of DNA Polymerase III of E. coli
Number of
subunits per
Subunit holoenzyme Mr of subunit Gene Function of subunit
 2 129,900 polC (dnaE) Polymerization activity
 2 27,500 dnaQ (mutD) 3
n5
Proofreading exonuclease Core polymerase

2 8,600 holE
2 71,100 dnaX Stable template binding;
core enzyme dimerization Clamp-loading () complex that
 1 47,500 dnaX* Clamp loader loads  subunits on lagging
 1 38,700 holA Clamp opener strand at each Okazaki fragment
 1 36,900 holB Clamp loader
1 16,600 holC Interaction with SSB

1 15,200 holD Interaction with  and
 4 40,600 dnaN DNA clamp required for
optimal processivity
*The  subunit is encoded by a portion of the gene for the  subunit, such that the amino-terminal 66% of the  subunit has
the same amino acid sequence as the  subunit. The  subunit is generated by a translational frameshifting mechanism (see
Box 27–1) that leads to premature translational termination.




another set of subunits, a clamp-loading complex, or

complex, consisting of five subunits of four different
types, 2
. The core polymerases are linked through
the  (tau) subunits. Two additional subunits,  (chi) and
 (psi), are bound to the clamp-loading complex. The
entire assembly of 13 protein subunits (nine different
types) is called DNA polymerase III* (Fig. 25–10a).
DNA polymerase III* can polymerize DNA, but with
a much lower processivity than one would expect for
the organized replication of an entire chromosome. The
necessary increase in processivity is provided by the addition
of the  subunits, four of which complete the DNA
polymerase III holoenzyme. The  subunits associate in
pairs to form donut-shaped structures that encircle the
DNA and act like clamps (Fig. 25–10b). Each dimer associates
with a core subassembly of polymerase III* (one
dimeric clamp per core subassembly) and slides along
the DNA as replication proceeds. The  sliding clamp
prevents the dissociation of DNA polymerase III from
DNA, dramatically increasing processivity—to greater
than 500,000 (Table 25–1).
DNA Replication Requires Many Enzymes
and Protein Factors
Replication in E. coli requires not just a single DNA
polymerase but 20 or more different enzymes and proteins,
each performing a specific task. The entire complex
has been termed the DNA replicase system or
replisome. The enzymatic complexity of replication reflects
the constraints imposed by the structure of DNA
and by the requirements for accuracy. The main classes
of replication enzymes are considered here in terms of
the problems they overcome.
Access to the DNA strands that are to act as templates
requires separation of the two parent strands.
This is generally accomplished by helicases, enzymes
that move along the DNA and separate the strands, using
chemical energy from ATP. Strand separation creates
topological stress in the helical DNA structure (see
Fig. 24–12), which is relieved by the action of topoisomerases.
The separated strands are stabilized by
DNA-binding proteins. As noted earlier, before DNA
polymerases can begin synthesizing DNA, primers must
be present on the template—generally short segments
25.1 DNA Replication 957

polymerase I
FIGURE 25–8 Large (Klenow) fragment of DNA polymerase I. This
polymerase is widely distributed in bacteria. The Klenow fragment,
produced by proteolytic treatment of the polymerase, retains the polymerization
and proofreading activities of the enzyme. The Klenow
fragment shown here is from the thermophilic bacterium Bacillus
stearothermophilus (PDB ID 3BDP). The active site for addition of nucleotides
is deep in the crevice at the far end of the bound DNA. The
dark blue strand is the template.
FIGURE 25–9 Nick translation. In this process, an RNA or DNA strand
paired to a DNA template is simultaneously degraded by the 5
n3

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.
End view
of RNA synthesized by enzymes known as primases.
Ultimately, the RNA primers are removed and replaced
by DNA; in E. coli, this is one of the many functions of
DNA polymerase I. After an RNA primer is removed and
the gap is filled in with DNA, a nick remains in the DNA
backbone in the form of a broken phosphodiester bond.
These nicks are sealed by DNA ligases. All these
processes require coordination and regulation, an interplay
best characterized in the E. coli system.
Replication of the E. coli Chromosome
Proceeds in Stages
The synthesis of a DNA molecule can be divided into
three stages: initiation, elongation, and termination,
distinguished both by the reactions taking place and by
the enzymes required. As you will find here and in the
next two chapters, synthesis of the major informationcontaining
biological polymers—DNAs, RNAs, and proteins—
can be understood in terms of these same three
stages, with the stages of each pathway having unique
characteristics. The events described below reflect information
derived primarily from in vitro experiments
using purified E. coli proteins, although the principles
are highly conserved in all replication systems.
Initiation The E. coli replication origin, oriC, consists
of 245 bp; it bears DNA sequence elements that are
highly conserved among bacterial replication origins.
The general arrangement of the conserved sequences is
958 Chapter 25 DNA Metabolism
t
b clamp
DnaB
helicase
t
b clamp
(open)
Core (aev)
d
g
d

FIGURE 25–10 DNA polymerase III. (a) Architecture of bacterial
DNA polymerase III. Two core domains, composed of subunits
, ,
and , are linked by a five-subunit  complex (also known as the
clamp-loading complex) with the composition 2. The  and 
subunits are encoded by the same gene. The  subunit is a shortened
version of ; the  subunit thus contains a domain identical to , along
with an additional segment that interacts with the core polymerase.
The other two subunits of DNA polymerase III*,  and (not shown),
also bind to the  complex. Two
clamps interact with the two-core
subassembly, each clamp a dimer of the
subunit. The complex interacts
with the DnaB helicase through the  subunit. (b) Two
subunits
of E. coli polymerase III form a circular clamp that surrounds the
DNA. The clamp slides along the DNA molecule, increasing the processivity
of the polymerase III holoenzyme to greater than 500,000 by
preventing its dissociation from the DNA. The end-on view shows the
two
subunits as gray and light-blue ribbon structures surrounding a
space-filling model of DNA. In the side view, surface contour models
of the
subunits (gray) surround a stick representation of a DNA double
helix (light and dark blue) (derived from PDB ID 2POL). Side view
(b)
(a)
illustrated in Figure 25–11. The key sequences of interest
here are two series of short repeats: three repeats
of a 13 bp sequence and four repeats of a 9 bp sequence.
At least nine different enzymes or proteins (summarized
in Table 25–3) participate in the initiation phase
of replication. They open the DNA helix at the origin
and establish a prepriming complex for subsequent reactions.
The crucial component in the initiation process
is the DnaA protein. A single complex of four to five
DnaA protein molecules binds to the four 9 bp repeats
in the origin (Fig. 25–12, step 1 ), then recognizes and
successively denatures the DNA in the region of the
three 13 bp repeats, which are rich in AUT pairs (step
2 ). This process requires ATP and the bacterial histonelike
protein HU. The DnaC protein then loads the
DnaB protein onto the unwound region. Two ringshaped
hexamers of DnaB, one loaded onto each DNA
strand, act as helicases, unwinding the DNA bidirectionally
and creating two potential replication forks. If
the E. coli single-stranded DNA–binding protein (SSB)
and DNA gyrase (DNA topoisomerase II) are now added
in vitro, thousands of base pairs are rapidly unwound
by the DnaB helicase, proceeding out from the origin.
Many molecules of SSB bind cooperatively to singlestranded
DNA, stabilizing the separated strands and
preventing renaturation while gyrase relieves the topological
stress produced by the DnaB helicase. When additional
replication proteins are included in the in vitro
system, the DNA unwinding mediated by DnaB is coupled
to replication, as described below.
Initiation is the only phase of DNA replication that
is known to be regulated, and it is regulated such that
replication occurs only once in each cell cycle. The
mechanism of regulation is not yet well understood, but
genetic and biochemical studies have provided a few
insights.
The timing of replication initiation is affected by
DNA methylation and interactions with the bacterial
plasma membrane. The oriC DNA is methylated by the
Dam methylase (Table 25–3), which methylates the N6
position of adenine within the palindromic sequence
(5
)GATC. (Dam is not a biochemical expletive; it stands
for DNA adenine methylation.) The oriC region of E. coli
is highly enriched in GATC sequences—it has 11 of them
in its 245 bp, whereas the average frequency of GATC in
the E. coli chromosome as a whole is 1 in 256 bp.
25.1 DNA Replication 959
Tandem array of
three 13 bp sequences
Binding sites for DnaA protein,
four 9 bp sequences
Consensus sequence
TTATCCACA
Consensus sequence
GATCTNTTNTTTT
FIGURE 25–11 Arrangement of sequences in the E. coli replication
origin, oriC. Although the repeated sequences (shaded in color) 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.
1
2
3
DnaB helicase
Priming and
replication
DnaB
DnaC
HU
DnaA
Supercoiled
template
Three 13 bp
repeats
Four 9 bp
repeats
oriC
 ATP
 ATP
 ATP
FIGURE 25–12 Model for initiation of replication at the E. coli origin,
oriC.
1 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 AUT-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 unwinds
the DNA in preparation for priming and DNA synthesis.
Immediately after replication, the DNA is hemimethylated:
the parent strands have methylated oriC
sequences but the newly synthesized strands do not. The
hemimethylated oriC sequences are now sequestered
for a period by interaction with the plasma membrane
(the mechanism is unknown). After a time, oriC is released
from the plasma membrane, and it must be fully
methylated by Dam methylase before it can again bind
DnaA. Regulation of initiation also involves the slow hydrolysis
of ATP by DnaA protein, which cycles the protein
between active (with bound ATP) and inactive (with
bound ADP) forms on a timescale of 20 to 40 minutes.
Elongation The elongation phase of replication includes
two distinct but related operations: leading strand synthesis
and lagging strand synthesis. Several enzymes at
the replication fork are important to the synthesis of both
strands. Parent DNA is first unwound by DNA helicases,
and the resulting topological stress is relieved by topoisomerases.
Each separated strand is then stabilized by
960 Chapter 25 DNA Metabolism
TABLE 25–3 Proteins Required to Initiate Replication at the E. coli Origin
Number of
Protein Mr subunits Function
DnaA protein 52,000 1 Recognizes ori sequence; opens duplex at specific sites in
origin
DnaB protein (helicase) 300,000 6* Unwinds DNA
DnaC protein 29,000 1 Required for DnaB binding at origin
HU 19,000 2 Histonelike protein; DNA-binding protein; stimulates initiation
Primase (DnaG protein) 60,000 1 Synthesizes RNA primers
Single-stranded DNA–binding
protein (SSB) 75,600 4* Binds single-stranded DNA
RNA polymerase 454,000 5 Facilitates DnaA activity
DNA gyrase (DNA topoisomerase II) 400,000 4 Relieves torsional strain generated by DNA unwinding
Dam methylase 32,000 1 Methylates (5
)GATC sequences at oriC
FIGURE 25–13 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.
5

3

5

3

5

3

Replication fork movement
Leading strand synthesis
(DNA polymerase III)
DnaB
helicase
DNA topoisomerase II
(DNA gyrase)
Lagging
strand
Lagging strand synthesis
(DNA polymerase III)
RNA SSB
primer
DNA
primase
(a)
(c)
(b)
RNA primer
from previous
Okazaki
fragment
*Subunits in these cases are identical.
SSB. From this point, synthesis of leading and lagging
strands is sharply different.
Leading strand synthesis, the more straightforward
of the two, begins with the synthesis by primase (DnaG
protein) of a short (10 to 60 nucleotide) RNA primer at
the replication origin. Deoxyribonucleotides are added
to this primer by DNA polymerase III. Leading strand
synthesis then proceeds continuously, keeping pace
with the unwinding of DNA at the replication fork.
Lagging strand synthesis, as we have noted, is accomplished
in short Okazaki fragments. First, an RNA
primer is synthesized by primase and, as in leading
strand synthesis, DNA polymerase III binds to the RNA
primer and adds deoxyribonucleotides (Fig. 25–13). On
this level, the synthesis of each Okazaki fragment seems
straightforward, but the reality is quite complex. The
complexity lies in the coordination of leading and lagging
strand synthesis: both strands are produced by a
single asymmetric DNA polymerase III dimer, which is
accomplished by looping the DNA of the lagging strand
as shown in Figure 25–14, bringing together the two
points of polymerization.
25.1 DNA Replication 961
DnaB
Core
Clamp-loading complex
with open b sliding clamp
Lagging strand
RNA primer
of previous
Okazaki
fragment
Leading
strand
(a) Continuous synthesis on the leading strand proceeds
as DNA is unwound by the DnaB helicase.
Primase
New
RNA
primer
Primer of previous
Okazaki fragment
approaches core
subunits
(b) DNA primase binds to DnaB, synthesizes
a new primer, then dissociates.
Primase
Discarded
b clamp
The next b clamp
is readied
New b clamp is loaded
onto new template primer
Synthesis of new
Okazaki fragment
is completed
(c)
New b clamp
(e)
(d)
FIGURE 25–14 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.
The synthesis of Okazaki fragments on the lagging
strand entails some elegant enzymatic choreography.
The DnaB helicase and DnaG primase constitute a functional
unit within the replication complex, the primosome.
DNA polymerase III uses one set of its core subunits
(the core polymerase) to synthesize the leading
strand continuously, while the other set of core subunits
cycles from one Okazaki fragment to the next on the
looped lagging strand. The DnaB helicase unwinds the
DNA at the replication fork (Fig. 25–14a) as it travels
along the lagging strand template in the 5
n3
direction.
DNA primase occasionally associates with DnaB
helicase and synthesizes a short RNA primer (Fig.
25–14b). A new  sliding clamp is then positioned at the
primer by the clamp-loading complex of DNA polymerase
III (Fig. 25–14c). When synthesis of an Okazaki
fragment has been completed, replication halts, and the
core subunits of DNA polymerase III dissociate from
their  sliding clamp (and from the completed Okazaki
fragment) and associate with the new clamp (Fig.
25–14d, e). This initiates synthesis of a new Okazaki
fragment. As noted earlier, the entire complex responsible
for coordinated DNA synthesis at a replication fork
is a replisome. The proteins acting at the replication
fork are summarized in Table 25–4.
The replisome promotes rapid DNA synthesis,
adding ~1,000 nucleotides/s to each strand (leading and
lagging). Once an Okazaki fragment has been completed,
its RNA primer is removed and replaced with
DNA by DNA polymerase I, and the remaining nick is
sealed by DNA ligase (Fig. 25–15).
DNA ligase catalyzes the formation of a phosphodiester
bond between a 3
hydroxyl at the end of one
DNA strand and a 5
phosphate at the end of another
strand. The phosphate must be activated by adenylylation.
DNA ligases isolated from viruses and eukaryotes
use ATP for this purpose. DNA ligases from bacteria are
unusual in that they generally use NAD—a cofactor
that normally functions in hydride transfer reactions
(see Fig. 13–15)—as the source of the AMP activating
group (Fig. 25–16). DNA ligase is another enzyme of
DNA metabolism that has become an important reagent
in recombinant DNA experiments (see Fig. 9–1).
Termination Eventually, the two replication forks of the
circular E. coli chromosome meet at a terminus region
containing multiple copies of a 20 bp sequence called
Ter (for terminus) (Fig. 25–17a). The Ter sequences are
arranged on the chromosome to create a sort of trap
that a replication fork can enter but cannot leave. The
Ter sequences function as binding sites for a protein
called Tus (terminus utilization substance). The Tus-Ter
complex can arrest a replication fork from only one direction.
Only one Tus-Ter complex functions per replication
cycle—the complex first encountered by either
962 Chapter 25 DNA Metabolism
TABLE 25–4 Proteins at the E. coli Replication Fork
Number of
Protein Mr subunits Function
SSB 75,600 4 Binding to single-stranded DNA
DnaB protein (helicase) 300,000 6 DNA unwinding; primosome constituent
Primase (DnaG protein) 60,000 1 RNA primer synthesis; primosome constituent
DNA polymerase III 791,500 17 New strand elongation
DNA polymerase I 103,000 1 Filling of gaps; excision of primers
DNA ligase 74,000 1 Ligation
DNA gyrase (DNA topoisomerase II) 400,000 4 Supercoiling
Modified from Kornberg, A. (1982) Supplement to DNA Replication, Table S11–2, W. H. Freeman and Company, New York.
3
5

5
3

Lagging
strand
dNTPs
rNMPs DNA polymerase I Nick
ATP (or NAD+)
AMP +PPi (or NMN)
DNA ligase
FIGURE 25–15 Final steps in the synthesis of lagging strand segments.
RNA primers in the lagging strand are removed by the 5
n3

exonuclease activity of DNA polymerase I and replaced with DNA by
the same enzyme. The remaining nick is sealed by DNA ligase. The
role of ATP or NAD is shown in Figure 25–16.
O
PPi (from ATP)
or
NMN (from NAD
)
Enzyme P O
O
O
Ribose Adenine
Enzyme
P
O
DNA ligase
OH O
Nick in DNA
Enzyme-AMP
NH3


O
P
OH O O
O O
P
O
O O
DNA ligase
P
O
O
O
Ribose Adenine
AMP
O P
O
O
Sealed DNA
Ribose Adenine
R O P O
O
O
Ribose Adenine
AMP from ATP (R  PPi)
or NAD
(R  NMN)
NH2


O O
Enzyme NH3


1 Adenylylation of
DNA ligase
2 Activation of
5 phosphate in
nick
5
3
3
5
3 Displacement of AMP seals nick
replication fork. Given that opposing replication forks
generally halt when they collide, Ter sequences do not
seem essential, but they may prevent overreplication by
one replication fork in the event that the other is delayed
or halted by an encounter with DNA damage or
some other obstacle.
So, when either replication fork encounters a functional
Tus-Ter complex, it halts; the other fork halts
when it meets the first (arrested) fork. The final few
hundred base pairs of DNA between these large protein
complexes are then replicated (by an as yet unknown
mechanism), completing two topologically interlinked
(catenated) circular chromosomes (Fig. 25–17b). DNA
circles linked in this way are known as catenanes. Separation
of the catenated circles in E. coli requires topoisomerase
IV (a type II topoisomerase). The separated
chromosomes then segregate into daughter cells at cell
division. The terminal phase of replication of other circular
chromosomes, including many of the DNA viruses
that infect eukaryotic cells, is similar.
Bacterial Replication Is Organized in Membrane-
Bound Replication Factories
The replication of a circular bacterial chromosome is
highly organized. Once bidirectional replication is initiated
at the origin, the two replisomes do not travel away
from each other along the DNA. Instead, the replisomes
are linked together and tethered to one point on the
bacterial inner membrane, and the DNA substrate is fed
through this “replication factory” (Fig. 25–18a). The
tethering point is at the center of the elongated bacterial
cell. After initiation, each of the two newly synthesized
replication origins is partitioned into one half of
25.1 DNA Replication 963
FIGURE 25–16 Mechanism of the DNA ligase reaction. In each of
the three steps, one phosphodiester bond is formed at the expense of
another. Steps
1 and
2 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
3 , the 3-
hydroxyl group attacks this phosphate and displaces AMP, producing a
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
1 .
(a)
Origin
Clockwise
fork
Counterclockwise
Clockwise fork trap
fork trap
Counterclockwise
fork
TerG
TerF
TerB TerC
TerA
TerD
TerB
Clockwise
fork
Counterclockwise
fork
completion
of replication
Catenated
chromosomes
Separated
chromosomes
(b)
DNA topoisomerase IV
the cell, and continuing replication extrudes each new
chromosome into that half (Fig. 25–18b). The elaborate
spatial organization of the newly replicated chromosomes
is orchestrated and maintained by many proteins,
including bacterial homologs of the SMC proteins and
topoisomerases (Chapter 24). Once replication is terminated,
the cell divides, and the chromosomes sequestered
in the two halves of the original cell are accurately
partitioned into the daughter cells. When
replication commences in the daughter cells, the origin
of replication is sequestered in new replication factories
formed at a point on the membrane at the center of the
cell, and the entire process is repeated.
Replication in Eukaryotic Cells Is More Complex
The DNA molecules in eukaryotic cells are considerably
larger than those in bacteria and are organized into complex
nucleoprotein structures (chromatin; p. 938). The
essential features of DNA replication are the same in
eukaryotes and prokaryotes, and many of the protein
complexes are functionally and structurally conserved.
However, some interesting variations on the general
principles discussed above promise new insights into the
regulation of replication and its link with the cell cycle.
Origins of replication, called autonomously replicating
sequences (ARS) or replicators, have been
identified and best studied in yeast. Yeast replicators
span ~150 bp and contain several essential conserved
sequences. About 400 replicators are distributed among
the 16 chromosomes in a haploid yeast genome. Initiation
of replication in all eukaryotes requires a multisubunit
protein, the origin recognition complex (ORC),
which binds to several sequences within the replicator.
ORC interacts with and is regulated by a number of
other proteins involved in control of the eukaryotic cell
cycle. Two other proteins, CDC6 (discovered in a screen
for genes affecting the cell division cycle) and CDT1
(Cdc10-dependent transcript 1), bind to ORC and mediate
the loading of a heterohexamer of minichromosome
maintenance proteins (MCM2 to MCM7). The
MCM complex is a ring-shaped replicative helicase, analogous
to the bacterial DnaB helicase. The CDC6 and
CDT1 proteins have a role comparable to that of the
bacterial DnaC protein, loading the MCM helicase onto
the DNA near the replication origin.
The rate of replication fork movement in eukaryotes
(~50 nucleotides/s) is only one-twentieth that observed
in E. coli. At this rate, replication of an average
human chromosome proceeding from a single origin
964 Chapter 25 DNA Metabolism
FIGURE 25–17 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 separated—except 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.
3
5

5
3

(a)
would take more than 500 hours. Replication of human
chromosomes in fact proceeds bidirectionally from
many origins, spaced 30,000 to 300,000 bp apart. Eukaryotic
chromosomes are almost always much larger
than bacterial chromosomes, so multiple origins are
probably a universal feature in eukaryotic cells.
Like bacteria, eukaryotes have several types of
DNA polymerases. Some have been linked to particular
functions, such as the replication of mitochondrial
DNA. The replication of nuclear chromosomes involves
DNA polymerase , in association with DNA polymerase
. DNA polymerase  is typically a multisubunit
enzyme with similar structure and properties in all
eukaryotic cells. One subunit has a primase activity, and
the largest subunit (Mr ~180,000) contains the polymerization
activity. However, this polymerase has no
proofreading 3
n5
exonuclease activity, making it unsuitable
for high-fidelity DNA replication. DNA polymerase
 is believed to function only in the synthesis
of short primers (containing either RNA or DNA) for
Okazaki fragments on the lagging strand. These primers
are then extended by the multisubunit DNA polymerase
. This enzyme is associated with and stimulated
by a protein called proliferating cell nuclear antigen
(PCNA; Mr 29,000), found in large amounts in the
nuclei of proliferating cells. The three-dimensional
structure of PCNA is remarkably similar to that of the
 subunit of E. coli DNA polymerase III (Fig. 25–10b),
although primary sequence homology is not evident.
PCNA has a function analogous to that of the  subunit,
forming a circular clamp that greatly enhances the
processivity of the polymerase. DNA polymerase  has
a 3
n5
proofreading exonuclease activity and appears
to carry out both leading and lagging strand synthesis
in a complex comparable to the dimeric bacterial DNA
polymerase III.
Yet another polymerase, DNA polymerase , replaces
DNA polymerase  in some situations, such as in
DNA repair. DNA polymerase  may also function at the
replication fork, perhaps playing a role analogous to that
of the bacterial DNA polymerase I, removing the primers
of Okazaki fragments on the lagging strand.
25.1 DNA Replication 965
Origin
Bacterium
Replisome
replication
begins
origins
separate
cell elongates
as replication
continues
chromosomes
separate
cells
divide
Terminator
(b)
Chromosome
FIGURE 25–18 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.
Many DNA viruses encode their own DNA polymerases,
and some of these have become targets for
pharmaceuticals. For example, the DNA polymerase of
the herpes simplex virus is inhibited by acyclovir, a compound
developed by Gertrude Elion (p. 876). Acyclovir
consists of guanine attached to an incomplete ribose
ring. It is phosphorylated by a virally encoded thymidine
kinase; acyclovir binds to this viral enzyme with an
affinity 200-fold greater than its binding to the cellular
thymidine kinase. This ensures that phosphorylation occurs
mainly in virus-infected cells. Cellular kinases convert
the resulting acyclo-GMP to acyclo-GTP, which is
both an inhibitor and a substrate of DNA polymerases,
and which competitively inhibits the herpes DNA polymerase
more strongly than cellular DNA polymerases.
Because it lacks a 3
hydroxyl, acyclo-GTP also acts as
a chain terminator when incorporated into DNA. Thus
viral replication is inhibited at several steps.
Two other protein complexes also function in eukaryotic
DNA replication. RPA (replication protein A)
is a eukaryotic single-stranded DNA–binding protein,
equivalent in function to the E. coli SSB protein. RFC
(replication factor C) is a clamp loader for PCNA and
facilitates the assembly of active replication complexes.
The subunits of the RFC complex have significant sequence
similarity to the subunits of the bacterial clamploading
() complex.
The termination of replication on linear eukaryotic
chromosomes involves the synthesis of special structures
called telomeres at the ends of each chromosome,
as discussed in the next chapter.
SUMMARY 25.1 DNA Replication
■ Replication of DNA occurs with very high
fidelity and at a designated time in the cell
cycle. Replication is semiconservative, each
strand acting as template for a new daughter
strand. It is carried out in three identifiable
phases: initiation, elongation, and termination.
The reaction starts at the origin and usually
proceeds bidirectionally.
■ DNA is synthesized in the 5
n3
direction by
DNA polymerases. At the replication fork, the
leading strand is synthesized continuously in
the same direction as replication fork
movement; the lagging strand is synthesized
discontinuously as Okazaki fragments, which
are subsequently ligated.
HN
N N
O
O
OH
H2N
N
■ The fidelity of DNA replication is maintained
by (1) base selection by the polymerase, (2) a
3
n5
proofreading exonuclease activity that is
part of most DNA polymerases, and (3) specific
repair systems for mismatches left behind after
replication.
■ Most cells have several DNA polymerases. In
E. coli, DNA polymerase III is the primary
replication enzyme. DNA polymerase I is
responsible for special functions during
replication, recombination, and repair.
■ Replication of the E. coli chromosome involves
many enzymes and protein factors organized in
replication factories, in which template DNA is
spooled through two replisomes tethered to the
bacterial plasma membrane.
■ Replication is similar in eukaryotic cells, but
eukaryotic chromosomes have many replication
origins.

Introduction

Permet la culture à grande échelle dans le but de produire divers molécules biologiques

Cependant la culture in vitro ne permet pas :

– la prolifération et la différenciation normale des cellules

– la reproduction de l’architecture tissulaire de l’organe

– les interactions cellules-cellules et cellules-matrice extracellulaire

I Lignées cellulaires

La culture de cellules primaires

Il s’agit de cellules issues du tissu d’un animal qui peuvent être dérivées de tissus normaux, d’embryons, de tumeurs. Ils peuvent provenir :

– d’un fragment de tissu (mélange de plusieurs types cellulaires)

– de cellules individualisées (désagrégation d’un fragment de tissu)

Méthodes de désagrégation du tissu :

– forces mécaniques : homogénéisation, sonication légère

– méthode enzymatique : trypsine, collagénase

– agents chélateurs : EDTA, EGTA

Enfin il faudra procéder au comptage des cellules et ensemencement (105-106 cellules / cm3).

La culture de cellules finies

Ce sont des cellules qui vont se diviser pendant un nombre donné de passage puis entrer en senescence (=ralentissement progressif de la prolifération cellulaire, puis la mort) et se diviser de moins en moins.

A noter que les lignées cellulaire finies provenant d’embryons vont se diviser plus que celles originaires d’un tissu adulte.

Exemple : W138 (fibroblastes humains) et MRC5 (tissu pulmonaire d’un fœtus)

La culture de cellules continues

Il s’agit ici de lignées cellulaires qui ont été transformées et immortalisées.

• La transformation

consiste en l’ introduction de changements dans une cellule conduisant à un phénotype de croissance et à l’immortalisation.

• Agents transformant :

– carcinogènes chimiques

– radiations ionisantes

– infection virale

  II Origine des lignées cellulaires

Organes à partir desquels les cellules peuvent être cultivées :

– système tégumentaire et muscle (mélanocytes, kératinocytes, muscles, cellules graisseuses)

–  tractus gastro-intestinal : épithéliums salivaire et intestinal, pancréas, foie

– système respiratoire : poumon, alvéole, bronche

– système reproducteur : utérus, cellules de Sertoli

– système endocrine : thyroïde, pancréas

– système ostéo-articulaire : chondrocytes, ostéoblastes

– système nerveux  : cellules gliales, ganglions

– système cardiovasculaire : myocytes, cellules endothéliales

– système hématopoïétique : progéniteurs, macrophages, lymphocytes

Ces lignées cellulaires proviennent principalement de l’homme et de la souris.

Il existe actuellement deux collections majeures de lignées cellulaires :

– ATCC : American Type Culture Collection

– ECACC : European Collection of Animal Cell Cultures

III Les types de lignées cellulaires

Cellules non adhérentes

Le sérum est décomplémenté par chauffage à 56°C pendant 30 min. Ce qui permet d’inactiver les protéines du complément qui pourraient nuire aux cellules. En effet le sérum peut-être une source majeure de contamination (virus, bactéries).

Les antibiotiques

Afin de prévenir d’éventuelles contaminations.