Chapitre 4 Réparation de l’ADNintranet.supbiotech.fr/courses/BIOLOGIE/document/2016-2017/Cours/... · → sauvegarde de l’information génétique -modifications de l’ADN car

Biotech 1 - 2016-2017 Réparation

BIOLOGIE– Biotech 1

MODULE BIOLOGIE MOLÉCULAIRE

Chapitre 4

Réparation de l’ADN


2

SOMMAIRE

1. Modifications de l’ADN 1.1. Mutations 1.2. Modifications physiologiques 1.3. Modifications induites

2. Méthodes de réparation 2.1. Réparation directe du dommage 2.2. Réparation des lésions d’un brin 2.3. Réparation des lésions simultanées des deux brins

3. Synthèse translésionnelle 3.1. Principe 3.2. Système SOS procaryote

4. Pathologies associées


3

1. MODIFICATIONS DE L’ADN

- systèmes de réparation (protéines, enzymes) : scrutent continuellement l’ADN

→ veillent sur l’intégrité de l’ADN

→ sauvegarde de l’information génétique

- modifications de l’ADN car constamment :

- divisions cellulaires : ADN polymérase fidèle, mais quelques mésappariements

- lésions de l’ADN, dues à divers agents : - internes (radicaux libres) - environnementaux (chaleur, UV, substances mutagènes)

- LÉSION : modification transitoire accidentelle ou provoquée qui aboutit à une anomalie de la structure chimique ou physique de l’ADN (multitude d’anomalies ≠)

- si la lésion n’est pas réparée avant la réplication de l’ADN : → risque d’insertion d’une base erronée face à la lésion = MUTATION → modification définitive et héréditaire

! NE PAS CONFONDRE LÉSION ET MUTATION !


4


réactifs génotoxiques accidents spontanés erreurs de réplication

184 Chapter 4: DNA, Chromosomes, and Genomes

The first striking feature of the human genome is how little of it (only a few percent) codes for proteins (Table 4–1 and Figure 4–16). It is also notable that nearly half of the chromosomal DNA is made up of mobile pieces of DNA that have gradually inserted themselves in the chromosomes over evolutionary time, multiplying like parasites in the genome (see Figure 4–62). We discuss these trans-posable elements in detail in later chapters.

A second notable feature of the human genome is the large average gene size—about 27,000 nucleotide pairs. As discussed above, a typical gene carries in its linear sequence of nucleotides the information for the linear sequence of the amino acids of a protein. Only about 1300 nucleotide pairs are required to encode a protein of average size (about 430 amino acids in humans). Most of the remain-ing sequence in a gene consists of long stretches of noncoding DNA that interrupt the relatively short segments of DNA that code for protein. As will be discussed in detail in Chapter 6, the coding sequences are called exons; the intervening (non-coding) sequences in genes are called introns (see Figure 4–15 and Table 4–1). The majority of human genes thus consist of a long string of alternating exons and introns, with most of the gene consisting of introns. In contrast, the majority of genes from organisms with concise genomes lack introns. This accounts for the much smaller size of their genes (about one-twentieth that of human genes), as well as for the much higher fraction of coding DNA in their chromosomes.

TABLE 4–1 Some Vital Statistics for the Human Genome

Human genome

DNA length 3.2 × 109 nucleotide pairs*

Number of genes coding for proteins Approximately 21,000

Largest gene coding for protein 2.4 × 106 nucleotide pairs

Mean size for protein-coding genes 27,000 nucleotide pairs

Smallest number of exons per gene 1

Largest number of exons per gene 178

Mean number of exons per gene 10.4

Largest exon size 17,106 nucleotide pairs

Mean exon size 145 nucleotide pairs

Number of noncoding RNA genes Approximately 9000**

Number of pseudogenes*** More than 20,000

Percentage of DNA sequence in exons (protein-coding sequences)

1.5%

Percentage of DNA in other highly conserved sequences****

3.5%

Percentage of DNA in high-copy-number repetitive elements

Approximately 50%

* The sequence of 2.85 billion nucleotides is known precisely (error rate of only about 1 in 100,000 nucleotides). The remaining DNA primarily consists of short sequences that are tandemly repeated many times over, with repeat numbers differing from one individual to the next. These highly repetitive blocks are hard to sequence accurately.** This number is only a very rough estimate.*** A pseudogene is a DNA sequence closely resembling that of a functional gene, but containing numerous mutations that prevent its proper expression or function. Most pseudogenes arise from the duplication of a functional gene followed by the accumulation of damaging mutations in one copy.**** These conserved functional regions include DNA encoding 5ʹ and 3ʹ UTRs (untranslated regions of mRNA), DNA specifying structural and functional RNAs, and DNA with conserved protein-binding sites.

(A)

(B)

MBoC6 m4.16/4.16

Figure 4–16 Scale of the human genome. If drawn with a 1 mm space between each nucleotide pair, as in (A), the human genome would extend 3200 km (approximately 2000 miles), far enough to stretch across the center of Africa, the site of our human origins (red line in B). At this scale, there would be, on average, a protein-coding gene every 150 m. An average gene would extend for 30 m, but the coding sequences in this gene would add up to only just over a meter.

DOMMAGES

détection des dommages

checkpoint du cycle cellulaire

systèmes de réparation

maladies génétiques, cancers,…

“by-pass” polymérases

mutations

ADN réparé apoptose


5


- plusieurs systèmes de réparation → spécialisation : chaque système répare un type de dommage

- utilisation de la structure double brin de l’ADN → le brin intact sert de matrice pour la réparation du brin endommagé → fidélité de la séquence réparée

- processus particulier à l’ADN → pas de réparation des ARNs ou des protéines

- modification sur 1 seul brin : 3 mécanismes selon nature et importance de la lésion - modifications sur les 2 brins : mécanismes plus complexes, moins fidèles

- dysfonctionnement des systèmes de réparation → pathologies


6

SOMMAIRE

1. Modifications de l’ADN 1.1. Mutations

1.1.1. Types de variants moléculaires 1.1.2. Conséquence d’une variation (mutation / polymorphisme)

1.2. Modifications physiologiques 1.2.1. Formes tautomères 1.2.2. Désaminations, dépurinations spontanées 1.2.3. Dommages oxydatifs de l’ADN 1.2.4. Erreurs de copie lors de la réplication

1.3. Modifications induites 1.3.1. Agents chimiques 1.3.2. Agents physiques

2. Méthodes de réparation 2.1. Réparation directe du dommage 2.2. Réparation des lésions d’un brin 2.3. Réparation des lésions simultanées des deux brins




7

1.1. Mutations


MODIFICATIONS GÉNÉTIQUES

- long terme → nécessaires pour variations génétiques et survie des espèces

mais… - court terme : stabilité génétique indispensable pour survie d’une cellule ou d’un

individu → équilibre à trouver

MUTATIONS

- modifications permanentes et transmissibles de la séquence d’ADN → peuvent modifier la séquence des acides aminés incorporés lors de la traduction

- spontanées et aléatoires : interviennent naturellement → principale source de diversité génétique

- induites : en réponse à une exposition à un agent mutagène qui augmente la fréquence des mutations


8

1.1. Mutations 1.1.1. Types de variants moléculaires


AVEC CHANGEMENT DE TAILLE DE LA MOLÉCULE D’ADN - insertion : addition d’une ou plusieurs paires de bases - délétion : perte d’une ou plusieurs paires de bases - inversion : renversement d’une séquence plus ou moins longue - duplication et amplification : copie X fois d’une séquence (de 2 nucléotides à plusieurs

centaines de kb)

SANS CHANGEMENT DE TAILLE DE LA MOLÉCULE D’ADN - substitution : remplacement d’un nucléotide par un autre (mutation ponctuelle)

- transition : échange d’une base purique contre une autre base purique (A ↔ G) ou d’une base pyrimidique contre une autre base pyrimidique (T ↔ C)

- transversion : échange d’une base purique contre une base pyrimidique (ou inversement)

A C

G T

transitions

transversions


9

1.1. Mutations 1.1.2. Conséquences d’une variation (mutation / polymorphisme)


selon localisation de la mutation → conséquences très différentesTraduction (4)

5’UTR E1 E2 E3 3’UTR promoteur

5’ 3’

ATG GT

TAA TGA TAG AATAAA

Les mutations (3)

Allongement de la protéine

Anomalies de la polyadénylation Absence d’épissage

AG

Epissage alternatif

Absence d’épissage

Mutations faux sens, non sens, épissage

Anomalie d’initiation de la traduction

Modifications quantitatives de l’expression génique

Il peut y avoir tout le long de la séquence des mutations muettes excepté pour ATG (M) et UGG (W)

Le code génétique (4)

E1 E2 E3 3’UTR5’UTRpromoteur

5’ 3’

ATG GT AG

TAA TGA TAG AATAAA

absence d’épissage

épissage alternatif

absence d’épissage

modifications quantitatives de l’expression génique

anomalie d’initiation de la traduction

mutations faux sens, non sens, épissage

allongement de la protéine

anomalies de la polyadénylation


10

1.1. Mutations 1.1.2. Conséquences d’une variation (mutation / polymorphisme)


- substitution : mutation ponctuelle → pas de modification du cadre de lecture de l’ARNm

(si contenue dans séquence codante d’un gène)

- mutation silencieuse : codon acide aminé → autre codon même acide aminé - mutation faux sens : codon acide aminé → codon autre acide aminé - mutation conservatrice : codon acide aminé → codon acide aminé du même groupe

(ex : Lys basique mutée en Arg basique) - mutation non sens : codon codant → codon stop - mutation frameshift : ajout ou délétion d’une ou plusieurs bases, déplaçant le cadre de

lecture

- “allèle muté” : la modification change le message exprimé - “allèle polymorphe” : la modification ne change pas le message exprimé

• Remarque : changement du cadre de lecture si insertion ou délétion ≠ multiple de 3 nucléotides


11

1.2. Modifications physiologiques 1.2.1. Formes tautomères


- existence de certaines bases azotées sous 2 formes tautomères → à l’origine d’appariements non classiques (ou mésappariements) : T-G (3 liaisons H) ou

C-A (2 liaisons H) → à pH 7 : formes cétone et amine majoritaires

HN

NH

O

O

N

NH

OH

O

thymine-céto thymine-énol(rare)

HN N

OH

O N

N NH

N

O

NH

H

H

guanine

thymine-énol

N

NN

NH

NH2

N

NHN

NH

NH

adénine-amineadénine-imine

(rare)

HN N

N

N NH

N

NH

HO

N

H

H

cytosine

adénine-imine


12

1.2. Modifications physiologiques 1.2.1. Formes tautomères


- correction des mésappariements lors de la relecture ou de la réparation post-réplicative - ou transmission aux molécules filles lors de la réplication :

A

A

A

C au lieu de T

C

AC C

G

G G

T

T T

paire normale

paire normale

paire anormale

paire anormale

Exemple : mésappariement dû à une transition T → C

- le brin porteur sert de matrice à la synthèse d’un nouveau brin - une seule réplication suffit pour aboutir à la substitution de TA en CG


13

1.2. Modifications physiologiques 1.2.2. Désaminations, dépurinations spontanées


modification assez fréquente → transition - cytosine → uracile - adénine → hypoxanthine - guanine → xanthine bases puriques

NH

N

NH2

O NH

NH

O

O

cytosine uracile

désamination

adénine hypoxanthine

N

NN

NH

NH2

N

NHN

NH

O

désamination présente en très petites quantité dans les acides nucléiques (anticodons des ARNt, base flottante)

nucléotide correspondant : inosine (I)

DÉSAMINATION OXYDATIVE SPONTANÉE (remplacement d’une amine par un carbonyle)


14



A

C

T

paire normale

paire normale

paire anormale

substitution CG en TA

G

désaminationspontanée de C

GU

GC

G GC C

U

AU A

- U peut être détectée et réparée - si absence de réparation → substitution de CG en TA en 2 réplications successives

DÉSAMINATION OXYDATIVE SPONTANÉE

appariement avec G appariement avec A

268 Chapter 5: DNA Replication, Repair, and Recombination

human cell loses about 18,000 purine bases (adenine and guanine) every day because their N-glycosyl linkages to deoxyribose hydrolyze, a spontaneous reac-tion called depurination. Similarly, a spontaneous deamination of cytosine to uracil in DNA occurs at a rate of about 100 bases per cell per day (Figure 5–38). DNA bases are also occasionally damaged by an encounter with reactive metab-olites produced in the cell, including reactive forms of oxygen and the high-en-ergy methyl donor S-adenosylmethionine, or by exposure to chemicals in the environment. Likewise, ultraviolet radiation from the sun can produce a covalent linkage between two adjacent pyrimidine bases in DNA to form, for example, thymine dimers (Figure 5–39). If left uncorrected when the DNA is replicated, most of these changes would be expected to lead either to the deletion of one or more base pairs or to a base-pair substitution in the daughter DNA chain (Figure 5–40). The mutations would then be propagated throughout subsequent cell gen-erations. Such a high rate of random changes in the DNA sequence would have disastrous consequences.

The DNA Double Helix Is Readily RepairedThe double-helical structure of DNA is ideally suited for repair because it carries two separate copies of all the genetic information—one in each of its two strands. Thus, when one strand is damaged, the complementary strand retains an intact copy of the same information, and this copy is generally used to restore the correct nucleotide sequences to the damaged strand.

An indication of the importance of a double-strand helix to the safe storage of genetic information is that all cells use it; only a few small viruses use single-strand DNA or RNA as their genetic material. The types of repair processes described in this section cannot operate on such nucleic acids, and once damaged, the chance of a permanent nucleotide change occurring in these single-strand genomes of viruses is thus very high. It seems that only organisms with tiny genomes (and therefore tiny targets for DNA damage) can afford to encode their genetic infor-mation in any molecule other than a DNA double helix.

Figure 5–38 Depurination and deamination. These reactions are two of the most frequent spontaneous chemical reactions that create serious DNA damage in cells. Depurination can release guanine (shown here), as well as adenine, from DNA. The major type of deamination reaction converts cytosine to an altered DNA base, uracil (shown here), but deamination occurs on other bases as well. These reactions normally take place in double-helical DNA; for convenience, only one strand is shown.

GUANINE

OO CH2P

O

O

O_

N

N N

NH

N

H

H

HO

GUANINE

OO CH2P

O

O

O_N

N N

NH

N

H

H

HO

DNAstrand

DNAstrand

H

OH

H2O

sugar phosphate afterdepurination

CYTOSINE URACIL

N

N

N

H H

H

H O

OO CH2P

O

O

O_

O

N

NHH

H O

OO CH2P

O

O

O_

H2O

NH3

DEAMINATION

DEPURINATION

MBoC6 m5.45/5.39


15



DÉSAMINATION OXYDATIVE SPONTANÉE

- mutations : répartition non équitable dans le génome - zones riches en CG (îlots CpG) : “points chauds” (“hotspot”) de mutation - chez l’Homme : C très souvent méthylées en position 5 → thymine suite à désamination → non reconnue comme erreur, pas de réparation

NH

N

NH2

O

5-méthylcytosine

NH

NH

O

O

désamination

thymine

- désamination oxydative aussi provoquée par agents chimiques (acide nitreux HNO2)

Remarque : pas de désamination possible sur la thymine


16



DÉPURINATION SPONTANÉE

- hydrolyse spontanée, dans les conditions physiologiques, de la liaison glycosidique (entre base et désoxyribose) → site abasique = site AP (apurique ou apyrimidique)

- dépurinations beaucoup plus fréquentes que dépyrimidations → environ 18 000 dépurinations / jour (6.109 bases puriques au total) → dépyrimidations : quelques centaines de bases


human cell loses about 18,000 purine bases (adenine and guanine) every day because their N-glycosyl linkages to deoxyribose hydrolyze, a spontaneous reac-tion called depurination. Similarly, a spontaneous deamination of cytosine to uracil in DNA occurs at a rate of about 100 bases per cell per day (Figure 5–38). DNA bases are also occasionally damaged by an encounter with reactive metab-olites produced in the cell, including reactive forms of oxygen and the high-en-ergy methyl donor S-adenosylmethionine, or by exposure to chemicals in the environment. Likewise, ultraviolet radiation from the sun can produce a covalent linkage between two adjacent pyrimidine bases in DNA to form, for example, thymine dimers (Figure 5–39). If left uncorrected when the DNA is replicated, most of these changes would be expected to lead either to the deletion of one or more base pairs or to a base-pair substitution in the daughter DNA chain (Figure 5–40). The mutations would then be propagated throughout subsequent cell gen-erations. Such a high rate of random changes in the DNA sequence would have disastrous consequences.

The DNA Double Helix Is Readily RepairedThe double-helical structure of DNA is ideally suited for repair because it carries two separate copies of all the genetic information—one in each of its two strands. Thus, when one strand is damaged, the complementary strand retains an intact copy of the same information, and this copy is generally used to restore the correct nucleotide sequences to the damaged strand.

An indication of the importance of a double-strand helix to the safe storage of genetic information is that all cells use it; only a few small viruses use single-strand DNA or RNA as their genetic material. The types of repair processes described in this section cannot operate on such nucleic acids, and once damaged, the chance of a permanent nucleotide change occurring in these single-strand genomes of viruses is thus very high. It seems that only organisms with tiny genomes (and therefore tiny targets for DNA damage) can afford to encode their genetic infor-mation in any molecule other than a DNA double helix.

Figure 5–38 Depurination and deamination. These reactions are two of the most frequent spontaneous chemical reactions that create serious DNA damage in cells. Depurination can release guanine (shown here), as well as adenine, from DNA. The major type of deamination reaction converts cytosine to an altered DNA base, uracil (shown here), but deamination occurs on other bases as well. These reactions normally take place in double-helical DNA; for convenience, only one strand is shown.

GUANINE

OO CH2P

O

O

O_

N

N N

NH

N

H

H

HO

GUANINE

OO CH2P

O

O

O_N

N N

NH

N

H

H

HO

DNAstrand

DNAstrand

H

OH

H2O

sugar phosphate afterdepurination

CYTOSINE URACIL

N

N

N

H H

H

H O

OO CH2P

O

O

O_

O

N

NHH

H O

OO CH2P

O

O

O_

H2O

NH3

DEAMINATION

DEPURINATION

MBoC6 m5.45/5.39

site abasique


17



- pas de réplication du site abasique → risque d’une dépurination = délétion d’un nucléotide

269

DNA Damage Can Be Removed by More Than One PathwayCells have multiple pathways to repair their DNA using different enzymes that act upon different kinds of lesions. Figure 5–41 shows two of the most common path-ways. In both, the damage is excised, the original DNA sequence is restored by a DNA polymerase that uses the undamaged strand as its template, and a remain-ing break in the double helix is sealed by DNA ligase (see Figure 5–12).

The two pathways differ in the way in which they remove the damage from DNA. The first pathway, called base excision repair, involves a battery of enzymes called DNA glycosylases, each of which can recognize a specific type of altered base in DNA and catalyze its hydrolytic removal. There are at least six types of these enzymes, including those that remove deaminated Cs, deaminated As, dif-ferent types of alkylated or oxidized bases, bases with opened rings, and bases in which a carbon–carbon double bond has been accidentally converted to a car-bon–carbon single bond. How is an altered base detected within the context of the double helix? A key step is an enzyme-mediated “flipping-out” of the altered nucleotide from the helix, which allows the DNA glycosylase to probe all faces of the base for damage (Figure 5–42). It is thought that these enzymes travel along DNA using base-flipping to evaluate the status of each base. Once an enzyme finds the damaged base that it recognizes, it removes that base from its sugar.

The “missing tooth” created by DNA glycosylase action is recognized by an enzyme called AP endonuclease (AP for apurinic or apyrimidinic, endo to signify that the nuclease cleaves within the polynucleotide chain), which cuts the phos-phodiester backbone, after which the resulting gap is repaired (see Figure 5–41A). Depurination, which is by far the most frequent type of damage suffered by DNA, also leaves a deoxyribose sugar with a missing base. Depurinations are directly repaired beginning with AP endonuclease, following the bottom half of the path-way in Figure 5–41A.

DNA REPAIR

Figure 5–39 The most common type of thymine dimer. This type of damage occurs in the DNA of cells exposed to ultraviolet irradiation (as in sunlight). A similar dimer will form between any two neighboring pyrimidine bases (C or T residues) in DNA.

H

O

O

CH3

CCN

N

O

O

CH3

P CCN

O

O

CH3

CCN

N

CC

H

O

O

CH3

CCN

N

CC

O

O

O

O

CC

CC

NH

H

H

H

H

H

MBoC6 m5.46/5.40

P

P

P

PP

PP

P

P

P

P

P

T

A

T

A

U

A

A

T

a G has beenchanged to an A

DNAREPLICATION

DNAREPLICATION

deaminated C

new strand

new strand

old strand

old strand

(A)

an A-T nucleotidepair has been deleted

mutated

unchanged

depurinated A

new strand

new strand

old strand

old strand

(B)

mutated

unchanged

MBoC6 m5.47/5.41

T

A

T

A

C

G

T

A

T

A

C

G T

T

A

T

A

C

G

A

T

T

A

T

A

C

G

A

T

T

A

T

A

U

G

A

T

Figure 5–40 How chemical modifications of nucleotides produce mutations. (A) Deamination of cytosine, if uncorrected, results in the substitution of one base for another when the DNA is replicated. As shown in Figure 5–38, deamination of cytosine produces uracil. Uracil differs from cytosine in its base-pairing properties and preferentially base-pairs with adenine. The DNA replication machinery therefore adds an adenine when it encounters a uracil on the template strand. (B) Depurination can lead to the loss of a nucleotide pair. When the replication machinery encounters a missing purine on the template strand, it may skip to the next complete nucleotide as illustrated here, thus producing a nucleotide deletion in the newly synthesized strand. Many other types of DNA damage (see Figure 5–37), if left uncorrected, also produce mutations when the DNA is replicated.

- dépurination → délétion d’une paire de bases - désamination → transition - formes tautomères → mésappariements

!


18

1.2. Modifications physiologiques 1.2.3. Dommages oxydatifs de l’ADN


- réaction des ROS avec molécules de l’organisme ROS (Reactive Oxygen Species) : radicaux libres particulièrement réactifs

→ dommages oxydatifs

- guanine particulièrement vulnérable aux dommages oxydatifs → 8-oxoguanine :

- lésion la plus fréquente - particulièrement mutagénique - appariement avec C OU A

HN

N NH

HN

H2N

O

O

8oxoGguanine

HN

N NH

N

H2N

O

NH

NH

O

O NH

NH

O

O

O

H

thymine 5-formyluracile

ribosedésoxyribose

O

OHOH

CH2OH OHO

HOH

CH2OH OH

ROS

ROS

ROS


19

1.2. Modifications physiologiques 1.2.3. Dommages oxydatifs de l’ADN


8-oxoG : appariement avec C ou A → transversion GC en TA après réplication → la plus commune des mutations associées au cancer

C

paire normale

transversion GC en TA

G ROS G8oxoG 8oxoG A

GC? A ou G

8oxoG ? A ou G

8oxoG T A

C C

C

G

GA ou C

A ou C


20

1.2. Modifications physiologiques 1.2.4. Erreurs de copie lors de la réplication


- erreurs de copies lors de la réplication → risques de mutations

- mutations spontanées dues aux erreurs : rares

- haute fidélité de la réplication grâce : - fonctions d’édition des ADN polymérases - systèmes de réparation

ADN polymérase nombre d’erreurs

sans fonction d’édition 1 erreur sur 10 000 nucléotides incorporés

avec fonction d’édition 1 erreur sur 106 à 107 nucléotides incorporés

avec systèmes de réparation 1 erreur sur 109 nucléotides incorporés

→ environ 3 mutations restantes après réplication complète du génome chez l’Homme


21

1.3. Modifications induites 1.3.1. Agents chimiques


- agents mutagènes exogènes → mésappariements ou perte de matériel génétique

- attaques d’agents chimiques sur les atomes nucléophiles des bases azotées (N7, N3, O2, O6) :

- alkylation : → transversions et transitions notamment sur O6 de G → souvent transversion de GC en TA (gaz moutarde)

- pontage interbrin (certains médicaments)

- agents intercalants : se placent entre deux plateaux de bases → erreurs de réplication (BET)

- incorporation d’analogues de bases (bromouracile, aminopurine) → erreurs de réplication


22

1.3. Modifications induites 1.3.2. Agents physiques


- radiations ionisantes → cassures simple brin ou double brin - rayonnements UV → formation de dimères pyrimidiques

269

DNA Damage Can Be Removed by More Than One PathwayCells have multiple pathways to repair their DNA using different enzymes that act upon different kinds of lesions. Figure 5–41 shows two of the most common path-ways. In both, the damage is excised, the original DNA sequence is restored by a DNA polymerase that uses the undamaged strand as its template, and a remain-ing break in the double helix is sealed by DNA ligase (see Figure 5–12).

The two pathways differ in the way in which they remove the damage from DNA. The first pathway, called base excision repair, involves a battery of enzymes called DNA glycosylases, each of which can recognize a specific type of altered base in DNA and catalyze its hydrolytic removal. There are at least six types of these enzymes, including those that remove deaminated Cs, deaminated As, dif-ferent types of alkylated or oxidized bases, bases with opened rings, and bases in which a carbon–carbon double bond has been accidentally converted to a car-bon–carbon single bond. How is an altered base detected within the context of the double helix? A key step is an enzyme-mediated “flipping-out” of the altered nucleotide from the helix, which allows the DNA glycosylase to probe all faces of the base for damage (Figure 5–42). It is thought that these enzymes travel along DNA using base-flipping to evaluate the status of each base. Once an enzyme finds the damaged base that it recognizes, it removes that base from its sugar.

The “missing tooth” created by DNA glycosylase action is recognized by an enzyme called AP endonuclease (AP for apurinic or apyrimidinic, endo to signify that the nuclease cleaves within the polynucleotide chain), which cuts the phos-phodiester backbone, after which the resulting gap is repaired (see Figure 5–41A). Depurination, which is by far the most frequent type of damage suffered by DNA, also leaves a deoxyribose sugar with a missing base. Depurinations are directly repaired beginning with AP endonuclease, following the bottom half of the path-way in Figure 5–41A.

DNA REPAIR

Figure 5–39 The most common type of thymine dimer. This type of damage occurs in the DNA of cells exposed to ultraviolet irradiation (as in sunlight). A similar dimer will form between any two neighboring pyrimidine bases (C or T residues) in DNA.

H

O

O

CH3

CCN

N

O

O

CH3

P CCN

O

O

CH3

CCN

N

CC

H

O

O

CH3

CCN

N

CC

O

O

O

O

CC

CC

NH

H

H

H

H

H

MBoC6 m5.46/5.40

P

P

P

PP

PP

P

P

P

P

P

T

A

T

A

U

A

A

T

a G has beenchanged to an A

DNAREPLICATION

DNAREPLICATION

deaminated C

new strand

new strand

old strand

old strand

(A)

an A-T nucleotidepair has been deleted

mutated

unchanged

depurinated A

new strand

new strand

old strand

old strand

(B)

mutated

unchanged

MBoC6 m5.47/5.41

T

A

T

A

C

G

T

A

T

A

C

G T

T

A

T

A

C

G

A

T

T

A

T

A

C

G

A

T

T

A

T

A

U

G

A

T

Figure 5–40 How chemical modifications of nucleotides produce mutations. (A) Deamination of cytosine, if uncorrected, results in the substitution of one base for another when the DNA is replicated. As shown in Figure 5–38, deamination of cytosine produces uracil. Uracil differs from cytosine in its base-pairing properties and preferentially base-pairs with adenine. The DNA replication machinery therefore adds an adenine when it encounters a uracil on the template strand. (B) Depurination can lead to the loss of a nucleotide pair. When the replication machinery encounters a missing purine on the template strand, it may skip to the next complete nucleotide as illustrated here, thus producing a nucleotide deletion in the newly synthesized strand. Many other types of DNA damage (see Figure 5–37), if left uncorrected, also produce mutations when the DNA is replicated.

notamment dimères de thymine : → liaison covalente intrabrin entre 2 thymines

adjacentes → distorsion de l’hélice, blocage de la polymérisation

(réplication et transcription)


23

RÉSUMÉ DES PRINCIPALES MODIFICATIONS

type de modification cause

base manquante dépurination par la chaleur ou par les acides (≃ 104 purines / jour chez l’Homme)

base altérée radiations ionisantes, agents alkylants

base incorrecte désamination spontanée (ex : cytosine → uracile ou adénine → hypoxanthine)

délétion - insertion agents intercalants

formation de dimères irradiation par les UV

coupure des brins radiations ionisantes, agents chimiques

cross-link entre les brins certains médicaments

formation de tautomères spontanée et transitoire



24


RÉSUMÉ DES PRINCIPALES MODIFICATIONS

U X

X

X

X

X(CH3)n

A G

X

X

XX

AA T

T

incorporation d’uracile

modification de base

alkylation

adduits volumineux

pontage intra-brin

5’

5’

coupure double-brins

pontage inter-brin

site abasique

coupure simple-brin

dimère de thymine

mésappariement

Principaux types de lésions de l’ADN


25

SOMMAIRE


2. Méthodes de réparation 2.1. Réparation directe du dommage

2.1.1. Réparation des méthylations de guanine 2.1.2. Réparation des dimères de thymine 2.1.3. Activité 3’ → 5’ exonucléasique de la polymérase

2.2. Réparation des lésions d’un brin 2.2.1. Réparation des mésappariements (Mismatch Repair MMR) 2.2.2. Système NER (Nucleotide Excision Repair) 2.2.3. Système BER (Base Excision Repair)

2.3. Réparation des lésions simultanées des deux brins 2.3.1. Jonction non homologue des extrémités 2.3.2. Système de recombinaison homologue (HR)




26

2. MÉTHODES DE RÉPARATION

RÉPARATION

correction immédiate

excision de base (BER)

excision de nucléotides (NER)

correction de mésappariements

(MMR)

réparation des cassures

synthèse translésionnelle

excisions courtes

excisions longues

réversion de la lésion

(transférases) recherche de l’information sur le brin complémentaire (excision)

recherche de l’information sur

une autre molécule

TOLÉRANCE

RÉPARATION SECONDAIRE


27

2.1. Réparation directe du dommage


2.1.1. Réparation des méthylations de guanine

- permet de réverser la lésion sans excision - rapide, immédiate - pas d’intervention d’ADN polymérase (pas de resynthèse d’ADN)

- réversion des alkylations par alkyltransférases : transfert du groupement alkyl de la base azotée sur fonction thiol d’une cystéine

- MGMT : déméthylation de la guanine en O6 - mutations de MGMT chez l’Homme : fréquence accrue de cancer (rôle suppresseur de

tumeur) - même type de réaction pour groupements éthyl

N

NHN

NH

O

NH2N

NN

NH NH2

OCH3

Cys SH

active inactive

Cys S CH3

O6-méthylguanine guanine

alkylation

méthylguanineméthyltransférase

MGMT


28

2.1. Réparation directe du dommage 2.1.2. Réparation des dimères de thymine


- réparation directe des dimères de thymine (dus à rayonnements UV) par photolyase - photolyase présente chez de nombreux organismes, mais pas mammifères - reconnaissance de la distorsion de l’hélice - activation par lumière bleue ou UV (λ > 300 nm) → transfert d’électrons et protons → réparation

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2.1.Réparation directe du dommage

La réparation directe permet de réverser la lésion sans excision. Elle est rapide et immédiate, ne nécessitant pas l’intervention d’une ADN polymérase pour la resynthèse d’ADN.

2.1.1.Réparation des méthylations de guanine

La réversion directe des alkylations est réalisées par des enzymes, les alkyltransférases, par transfert du groupement alkyl de la base azotée sur la fonction thiol d’une cystéine.

!

La méthylguanine méthyltransférase MGMT catalyse la déméthylation de la guanine en O6. Le même type de réaction existe pour les groupements éthyl. Chez l’homme, les mutations de MGMT sont associées à une fréquence accrue de cancer (rôle suppresseur de tumeur).

2.1.2.Réparation des dimères de thymine

Les dimères de thymine provoqués par les rayonnements UV peuvent être réparés directement par l’action d’une photolyase. Cette photolyase est présente dans de nombreux organismes vivants mais n’est pas retrouvée chez les mammifères. La photolyase reconnaît la distorsion de l’hélice provoquée par le dimère de thymine. Activée par de la lumière bleue ou ultraviolette (λ > 300 nm), elle catalyse un transfert d’électron et de proton permettant de réparer le dimère.

!

2.1.3.Activité 3’ → 5’ exonucléasique de la polymérase

L’ADN polymérase possède une activité 3’ → 5’ exonucléasique qui lui permet de vérifier l’appariement du dernier nucléotide incorporé et de corriger ses éventuelles erreurs lors de la réplication (voir chapitre sur la réplication).

N

NHN

NH

O

NH2N

NN

NH NH2

OCH3

Cys SH

active inactive

Cys S CH3

O6-méthylguanine guanine

alkylation

méthylguanineméthyltransférase

MGMT

Correction immédiate3. correction immédiate

erreurs d’incorporations§1/104 pb vs 1/108 pb final

arrêt de la polyméraseactivité 3’-5’ exonucléase reprise de synthèse 5’- 3’

photolyases

réversion directe par des alkyltransférases (ex: O6-méthylguanine- DNA méthyl transférase = MGMT)

activité 3’-5’ exonucléase des polymérases

ADN avec dimère de T liaison de la photolyase au dimère

absorption de la lumière réversion et correction de l’erreur, largage de la lyase

Réparation -! -11


29

2.1. Réparation directe du dommage 2.1.3. Activité 3’ → 5’ exonucléasique de la polymérase



There is an error frequency of about one mistake for every 104 polymerization events both in RNA synthesis and in the separate process of translating mRNA sequences into protein sequences. This error rate is over 100,000 times greater than that in DNA replication, where, as we have seen, a series of proofreading processes makes the process unusually accurate (Table 5–1).

Only DNA Replication in the 5ʹ-to-3ʹ Direction Allows Efficient Error CorrectionThe need for accuracy probably explains why DNA replication occurs only in the 5ʹ-to-3ʹ direction. If there were a DNA polymerase that added deoxyribonucleo-side triphosphates in the 3ʹ-to-5ʹ direction, the growing 5ʹ end of the chain, rather than the incoming mononucleotide, would have to provide the activating triphos-phate needed for the covalent linkage. In this case, the mistakes in polymeriza-tion could not be simply hydrolyzed away, because the bare 5ʹ end of the chain thus created would immediately terminate DNA synthesis (see Figure 5–3). It is therefore possible to correct a mismatched base only if it has been added to the 3ʹ end of a DNA chain. Although the backstitching mechanism for DNA replica-tion seems complex, it preserves the 5ʹ-to-3ʹ direction of polymerization that is required for exonucleolytic proofreading.

Despite these safeguards against DNA replication errors, DNA polymerases occasionally make mistakes. However, as we shall see later, cells have yet another

5′

5′3′

POLYMERIZING EDITING

PE EP

templatestrand

newlysynthesized

DNA

MBoC6 m5.09/5.09

Figure 5–9 Editing by DNA polymerase. DNA polymerase complexed with the DNA template in the polymerizing mode (left) and the editing mode (right). The catalytic sites for the exonucleolytic (E) and the polymerization (P) reactions are indicated. In the editing mode, the newly synthesized DNA transiently unpairs from the template and enters the editing site where the most recently added nucleotide is catalytically removed.

TABLE 5–1 The Three Steps That Give Rise to High-Fidelity DNA Synthesis

Replication step Errors per nucleotide added

5ʹ → 3ʹ polymerization 1 in 105

3ʹ → 5ʹ exonucleolytic proofreading 1 in 102

Strand-directed mismatch repair 1 in 103

Combined 1 in 1010

The third step, strand-directed mismatch repair, is described later in this chapter. For the polymerization step, “errors per nucleotide added” describes the probability that an incorrect nucleotide will be added to the growing chain. For the other two steps, “errors per nucleotide added” describes the probability that an error will not be corrected. Each step therefore reduces the chance of a final error by the factor shown.

- ADN polymérase : enzyme “auto-correctrice” (fonction d’édition)

- si incorporation d’un nucléotide non complémentaire → arrêt réaction polymérisation, redirection du brin vers le site exonucléasique → nucléotide incorrect retiré grâce à l’activité exonucléasique 3’-5’

= clivage de la dernière liaison phosphodiester, de 3’ vers 5’ → reprise de la polymérisation

cf chapitre Réplication de l’ADN


30

2.2. Réparation des lésions d’un brin


- l’information perdue sur un brin existe sur l’autre !

- principe général :

- RECONNAISSANCE DE LA LÉSION - COUPURE - RESYNTHÈSE - LIGATION

→ RESTAURATION

reconnaissance de la lésion


30

2.2. Réparation des lésions d’un brin


- l’information perdue sur un brin existe sur l’autre !

- principe général :

- RECONNAISSANCE DE LA LÉSION - COUPURE - RESYNTHÈSE - LIGATION

→ RESTAURATION

251

and cells deficient in mismatch proofreading therefore have a greatly enhanced chance of becoming cancerous. Fortunately, most of us inherit two good cop-ies of each gene that encodes a mismatch proofreading protein; this protects us, because it is highly unlikely for both copies to become mutated in the same cell.

DNA Topoisomerases Prevent DNA Tangling During ReplicationAs a replication fork moves along double-strand DNA, it creates what has been called the “winding problem.” The two parental strands, which are wound around each other, must be unwound and separated for replication to occur. For every 10 nucleotide pairs replicated at the fork, one complete turn of the parental double helix must be unwound. In principle, this unwinding can be achieved by rapidly rotating the entire chromosome ahead of a moving fork; however, this is energet-ically highly unfavorable (particularly for long chromosomes) and, instead, the DNA in front of a replication fork becomes overwound (Figure 5–20). The over-winding, in turn, is continually relieved by proteins known as DNA topoisomer-ases.

A DNA topoisomerase can be viewed as a reversible nuclease that adds itself covalently to a DNA backbone phosphate, thereby breaking a phosphodiester bond in a DNA strand. This reaction is reversible, and the phosphodiester bond re-forms as the protein leaves.

One type of topoisomerase, called topoisomerase I, produces a transient sin-gle-strand break; this break in the phosphodiester backbone allows the two sec-tions of DNA helix on either side of the nick to rotate freely relative to each other, using the phosphodiester bond in the strand opposite the nick as a swivel point (Figure 5–21). Any tension in the DNA helix will drive this rotation in the direction that relieves the tension. As a result, DNA replication can occur with the rotation of only a short length of helix—the part just ahead of the fork. Because the cova-lent linkage that joins the DNA topoisomerase protein to a DNA phosphate retains

DNA REPLICATION MECHANISMS

error in newlymade strand

BINDING OF MISMATCHPROOFREADING PROTEINS

DNA SCANNING DETECTSNICK IN NEW DNA STRAND

STRAND REMOVAL

REPAIR DNA SYNTHESIS

(A) (B)

MutS MutL

MBoC6 m5.20/5.19

Figure 5–19 Strand-directed mismatch repair. (A) The two proteins shown are present in both bacteria and eukaryotic cells: MutS binds specifically to a mismatched base pair, while MutL scans the nearby DNA for a nick. Once MutL finds a nick, it triggers the degradation of the nicked strand all the way back through the mismatch. Because nicks are largely confined to newly replicated strands in eukaryotes, replication errors are selectively removed. In bacteria, an additional protein in the complex (MutH) nicks unmethylated (and therefore newly replicated) GATC sequences, thereby beginning the process illustrated here. In eukaryotes, MutL contains a DNA nicking activity that aids in the removal of the damaged strand. (B) The structure of the MutS protein bound to a DNA mismatch. This protein is a dimer, which grips the DNA double helix as shown, kinking the DNA at the mismatched base pair. It seems that the MutS protein scans the DNA for mismatches by testing for sites that can be readily kinked, which are those with an abnormal base pair. (PDB code: 1EWQ.)

3′3′

5′ 5′

(A) (B)

if the DNA cannot rapidlyrotate, torsional stresswill build up lagging-strand

template

leading-strandtemplate

MBoC6 m5.21/5.20

Figure 5–20 The “winding problem” that arises during DNA replication. (A) For a bacterial replication fork moving at 500 nucleotides per second, the parental DNA helix ahead of the fork must rotate at 50 revolutions per second. (B) If the ends of the DNA double helix remain fixed (or difficult to rotate), tension builds up in front of the replication fork as it becomes overwound. Some of this tension can be taken up by supercoiling, whereby the DNA double helix twists around itself (see Figure 6–19). However, if the tension continues to build up, the replication fork will eventually stop because further unwinding requires more energy than the helicase can provide. Note that in (A), the dotted line represents about 20 turns of DNA.

251

and cells deficient in mismatch proofreading therefore have a greatly enhanced chance of becoming cancerous. Fortunately, most of us inherit two good cop-ies of each gene that encodes a mismatch proofreading protein; this protects us, because it is highly unlikely for both copies to become mutated in the same cell.

DNA Topoisomerases Prevent DNA Tangling During ReplicationAs a replication fork moves along double-strand DNA, it creates what has been called the “winding problem.” The two parental strands, which are wound around each other, must be unwound and separated for replication to occur. For every 10 nucleotide pairs replicated at the fork, one complete turn of the parental double helix must be unwound. In principle, this unwinding can be achieved by rapidly rotating the entire chromosome ahead of a moving fork; however, this is energet-ically highly unfavorable (particularly for long chromosomes) and, instead, the DNA in front of a replication fork becomes overwound (Figure 5–20). The over-winding, in turn, is continually relieved by proteins known as DNA topoisomer-ases.

A DNA topoisomerase can be viewed as a reversible nuclease that adds itself covalently to a DNA backbone phosphate, thereby breaking a phosphodiester bond in a DNA strand. This reaction is reversible, and the phosphodiester bond re-forms as the protein leaves.

One type of topoisomerase, called topoisomerase I, produces a transient sin-gle-strand break; this break in the phosphodiester backbone allows the two sec-tions of DNA helix on either side of the nick to rotate freely relative to each other, using the phosphodiester bond in the strand opposite the nick as a swivel point (Figure 5–21). Any tension in the DNA helix will drive this rotation in the direction that relieves the tension. As a result, DNA replication can occur with the rotation of only a short length of helix—the part just ahead of the fork. Because the cova-lent linkage that joins the DNA topoisomerase protein to a DNA phosphate retains

DNA REPLICATION MECHANISMS

error in newlymade strand

BINDING OF MISMATCHPROOFREADING PROTEINS

DNA SCANNING DETECTSNICK IN NEW DNA STRAND

STRAND REMOVAL

REPAIR DNA SYNTHESIS

(A) (B)

MutS MutL

MBoC6 m5.20/5.19

Figure 5–19 Strand-directed mismatch repair. (A) The two proteins shown are present in both bacteria and eukaryotic cells: MutS binds specifically to a mismatched base pair, while MutL scans the nearby DNA for a nick. Once MutL finds a nick, it triggers the degradation of the nicked strand all the way back through the mismatch. Because nicks are largely confined to newly replicated strands in eukaryotes, replication errors are selectively removed. In bacteria, an additional protein in the complex (MutH) nicks unmethylated (and therefore newly replicated) GATC sequences, thereby beginning the process illustrated here. In eukaryotes, MutL contains a DNA nicking activity that aids in the removal of the damaged strand. (B) The structure of the MutS protein bound to a DNA mismatch. This protein is a dimer, which grips the DNA double helix as shown, kinking the DNA at the mismatched base pair. It seems that the MutS protein scans the DNA for mismatches by testing for sites that can be readily kinked, which are those with an abnormal base pair. (PDB code: 1EWQ.)

3′3′

5′ 5′

(A) (B)

if the DNA cannot rapidlyrotate, torsional stresswill build up lagging-strand

template

leading-strandtemplate

MBoC6 m5.21/5.20

Figure 5–20 The “winding problem” that arises during DNA replication. (A) For a bacterial replication fork moving at 500 nucleotides per second, the parental DNA helix ahead of the fork must rotate at 50 revolutions per second. (B) If the ends of the DNA double helix remain fixed (or difficult to rotate), tension builds up in front of the replication fork as it becomes overwound. Some of this tension can be taken up by supercoiling, whereby the DNA double helix twists around itself (see Figure 6–19). However, if the tension continues to build up, the replication fork will eventually stop because further unwinding requires more energy than the helicase can provide. Note that in (A), the dotted line represents about 20 turns of DNA.

reconnaissance de la lésion

coupure (± longue)

resynthèse et ligation


31

2.2. Réparation des lésions d’un brin 2.2.1. Réparation des mésappariements (Mismatch Repair MMR)


- réparation très précoce après réplication - mécanismes très conservés, des bactéries à l’Homme - chez E.coli, protéines Mut

- réparation ciblée du brin néoformé (utilisation de cassures simple brin plus fréquentes que dans brin parental)

- équivalents des gènes mut chez l’Homme : MSH2, MLH1, MSH6 - mutations sur gène MSH2 (équivalent de mutS) → cancers colorectaux

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Ce sont des réparations secondaires qui interviennent rapidement après la réplication : la lésion est excisée puis la brèche est resynthétisée par l’ADN polymérase à partir des informations portées par le brin intact.

2.2.1.Réparation des mésappariements (Mismatch Repair MMR)

La réparation des mésappariements a lieu très précocement après la réplication. Le mécanisme est conservé depuis les bactéries jusqu’à l’homme. Chez E. coli, cette réparation est assurée par les protéines Mut :

!

- MutS repère les distorsions de l’hélice d’ADN engendrées par un mésappariement - MutL permet l’interaction entre MutS et MutH - MutH est une endonucléase : elle identifie le brin néoformé et le brin parental, forme une boucle et

clive le brin néoformé à quelques bases du mésappariement - une exonucléase digère le brin néoformé au niveau de la boucle en éliminant le nucléotide

responsable du mauvais appariement - l’ADN polymérase III répare le brin néoformé par resynthèse d’ADN en utilisant le brin parental

comme matrice - une ADN ligase referme la brèche.

La réparation est ciblée vers le brin néoformé : elle utilise des cassures simple brin qui sont plus fréquentes dans le brin néoformé que dans le brin parental. Chez l’homme, ce sont les équivalents des gènes mut de E. coli qui sont impliqués : MSH2, MLH1 et MSH6. Des mutations de MSH2 (équivalent de mutS) sur les deux allèles sont à l’origine de cancers colorectaux.

2.2.2.Système NER (Nucleotide Excision Repair)

Le mécanisme NER est un mécanisme de réparation très fidèle des lésions volumineuses comme les dimères pyrimidiques provoqués par les rayonnements ultraviolets. Le dommage à l’ADN est d’abord repéré, puis l’ADN est clivé de part et d’autre du dommage (fragment de 12 nucléotides). Une hélicase déroule la double hélice d’ADN pour libérer le fragment clivé. Enfin, une ADN polymérase resynthétise le fragment manquant d’après la séquence du brin intact et une ligase referme le brin d’ADN.

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détection du mésappariement (MutS)

identification et clivage du brin

néoformé (MutH)

digestion du brin néoformé (nucléase)

réparation par synthèse d’ADN

Mut S Mut L

Mut H

mésappariement (erreur dans le nouveau brin)

ADN polymérase + ligase

Réparation -! -12

- MutS repère les distorsions de l’hélice engendrées par un mésappariement

- Mut L permet l’interaction entre MutS et Mut H - MutH = endonucléase → identifie le brin néoformé et le brin parental → forme une boucle et clive le brin néoformé à

quelques bases du mésappariement - digest ion du b r i n néo fo rmé pa r une

exonucléase, élimination du nucléotide à l’origine du mésappariement

- resynthèse d’ADN par ADN polymérase III, (matrice = brin parental)

- fermeture de la brèche par ADN ligase


32

2.2. Réparation des lésions d’un brin 2.2.2. Système NER (Nucleotide Excision Repair)


- réparation très fidèle des lésions volumineuses (dimères de pyrimidines provoqués par rayonnements UV)

- repérage de la distorsion - ADN clivé de part et d’autre du dommage

(fragment de 12 nucléotides) par une nucléase - hélicase déroule la double hélice et libère le

fragment clivé - resynthèse d’ADN par ADN polymérase III,

(matrice = brin intact) - fermeture de la brèche par ADN ligase


The second major repair pathway is called nucleotide excision repair. This mechanism can repair the damage caused by almost any large change in the structure of the DNA double helix. Such “bulky lesions” include those created by the covalent reaction of DNA bases with large hydrocarbons (such as the carcino-gen benzopyrene, found in tobacco smoke, coal tar, and diesel exhaust), as well as the various pyrimidine dimers (T-T, T-C, and C-C) caused by sunlight. In this path-way, a large multienzyme complex scans the DNA for a distortion in the double helix, rather than for a specific base change. Once it finds a lesion, it cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion, and a DNA helicase peels away the single-strand oligonucleotide containing the lesion. The large gap produced in the DNA helix is then repaired by DNA poly-merase and DNA ligase (see Figure 5–41B).

An alternative to base and nucleotide excision repair processes is direct chemi-cal reversal of DNA damage, and this strategy is selectively employed for the rapid

hydrogen-bondedbase pairs

G C T U A T C C

C G A G T A G G

G C T A T C C

C G A G T A G G

G C T A T C C

C G A G T A G G

UURACIL DNAGLYCOSYLASE

AP ENDONUCLEASE AND PHOSPHODIESTERASEREMOVE SUGAR PHOSPHATE

DNA helixwith missingbase

DNA POLYMERASE ADDS NEWNUCLEOTIDE, DNA LIGASESEALS NICK

MBoC6 m5.48/5.42

deaminated C

DNA helixwith single-nucleotide gap

G A T G C C A G A T G A T A C C

C T A C G G T C T A C T A T G G

pyrimidine dimer



C T A

C G G T C T A C T A T G

G

5'

3'

5'

3'

EXCISIONNUCLEASE

DNAHELICASE

DNA POLYMERASEPLUS DNA LIGASE

DNA helixwith 12-nucleotide gap

(A) BASE EXCISION REPAIR (B) NUCLEOTIDE EXCISION REPAIR





G C T C A T C C

C G A G T A G G

Figure 5– 41 A comparison of two major DNA repair pathways. (A) Base excision repair. This pathway starts with a DNA glycosylase. Here, the enzyme uracil DNA glycosylase removes an accidentally deaminated cytosine in DNA. After the action of this glycosylase (or another DNA glycosylase that recognizes a different kind of damage), the sugar phosphate with the missing base is cut out by the sequential action of AP endonuclease and a phosphodiesterase. (These same enzymes begin the repair of depurinated sites directly.) The gap of a single nucleotide is then filled by DNA polymerase and DNA ligase. The net result is that the U that was created by accidental deamination is restored to a C. AP endonuclease is so-named because it recognizes any site in the DNA helix that contains a deoxyribose sugar with a missing base; such sites can arise either by the loss of a purine (apurinic sites) or by the loss of a pyrimidine (apyrimidinic sites). (B) Nucleotide excision repair. In bacteria, after a multienzyme complex has recognized a lesion such as a pyrimidine dimer (see Figure 5–39), one cut is made on each side of the lesion, and an associated DNA helicase then removes the entire portion of the damaged strand. The excision repair machinery in bacteria leaves the gap of 12 nucleotides shown. In humans, once the damaged DNA is recognized, a helicase is recruited to unwind the DNA duplex locally. Next, the excision nuclease enters and cleaves on either side of the damage, leaving a gap of about 30 nucleotides. The nucleotide excision repair machinery in both bacteria and humans can recognize and repair many different types of DNA damage.


33

2.2. Réparation des lésions d’un brin 2.2.2. Système NER (Nucleotide Excision Repair)


- bactéries : protéines Uvr (A, B, C, D) - Homme : protéines XP (A, B, C, D, F, G) - mutations dans les gènes codant protéines XP → maladies dont Xeroderma

Pigmentosum

XERODERMA PIGMENTOSUM

- maladie très rare (prévalence ≃ 1/106 en Europe et USA) - transmise génétiquement (mode autosomique récessif) - hypersensibilité aux rayons UV, plus rarement désordres neurologiques - pas de réparation des lésions de l’ADN causées par les UV → défaut de réparation par excision de nucléotides → tumeurs de la peau dans les zones exposées → espérance de vie très réduite


34

2.2. Réparation des lésions d’un brin 2.2.3. Système BER (Base Excision Repair)


271

removal of certain highly mutagenic or cytotoxic lesions. For example, the alkyla-tion lesion O6-methylguanine has its methyl group removed by direct transfer to a cysteine residue in the repair protein itself, which is destroyed in the reaction. In another example, methyl groups in the alkylation lesions 1-methyladenine and 3-methylcytosine are “burnt off” by an iron-dependent demethylase, with release of formaldehyde from the methylated DNA and regeneration of the native base.

Coupling Nucleotide Excision Repair to Transcription Ensures That the Cell’s Most Important DNA Is Efficiently Repaired All of a cell’s DNA is under constant surveillance for damage, and the repair mech-anisms we have described act on all parts of the genome. However, cells have a way of directing DNA repair to the DNA sequences that are most urgently needed. They do this by linking RNA polymerase, the enzyme that transcribes DNA into RNA as the first step in gene expression, to the nucleotide excision repair pathway. As discussed above, this repair system can correct many different types of DNA damage. RNA polymerase stalls at DNA lesions and, through the use of coupling proteins, directs the excision repair machinery to these sites. In bacteria, where genes are relatively short, the stalled RNA polymerase can be dissociated from the DNA; the DNA is repaired, and the gene is transcribed again from the beginning. In eukaryotes, where genes can be enormously long, a more complex reaction is used to “back up” the RNA polymerase, repair the damage, and then restart the polymerase.

The importance of transcription-coupled excision repair is seen in people with Cockayne syndrome, which is caused by a defect in this coupling. These indi-viduals suffer from growth retardation, skeletal abnormalities, progressive neural retardation, and severe sensitivity to sunlight. Most of these problems are thought to arise from RNA polymerase molecules that become permanently stalled at sites of DNA damage that lie in important genes.

The Chemistry of the DNA Bases Facilitates Damage DetectionThe DNA double helix seems optimal for repair. As noted above, it contains a backup copy of all genetic information. Equally importantly, the nature of the four bases in DNA makes the distinction between undamaged and damaged bases very clear. For example, every possible deamination event in DNA yields an “unnatural” base, which can be directly recognized and removed by a specific DNA glycosylase. Hypoxanthine, for example, is the simplest purine base capable of pairing specifically with C, but hypoxanthine is the direct deamination prod-uct of A (Figure 5–43A). The addition of a second amino group to hypoxanthine

(A) (B)

MBoC6 m5.49/5.43

Figure 5–42 The recognition of an unusual nucleotide in DNA by base-flipping. The DNA glycosylase family of enzymes recognizes specific inappropriate bases in the conformation shown. Each of these enzymes cleaves the glycosyl bond that connects a particular recognized base (yellow) to the backbone sugar, removing it from the DNA. (A) Stick model; (B) space-filling model.

DNA REPAIR

- reconnaissance nucléotide altéré (hypoxanthine, uracile, xanthine, formés par désamination oxydative de A, C et G respectivement) par ADN glysosylases spécifiques

- basculement du nucléotide altéré vers l’extérieur de l’hélice


35


The second major repair pathway is called nucleotide excision repair. This mechanism can repair the damage caused by almost any large change in the structure of the DNA double helix. Such “bulky lesions” include those created by the covalent reaction of DNA bases with large hydrocarbons (such as the carcino-gen benzopyrene, found in tobacco smoke, coal tar, and diesel exhaust), as well as the various pyrimidine dimers (T-T, T-C, and C-C) caused by sunlight. In this path-way, a large multienzyme complex scans the DNA for a distortion in the double helix, rather than for a specific base change. Once it finds a lesion, it cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion, and a DNA helicase peels away the single-strand oligonucleotide containing the lesion. The large gap produced in the DNA helix is then repaired by DNA poly-merase and DNA ligase (see Figure 5–41B).

An alternative to base and nucleotide excision repair processes is direct chemi-cal reversal of DNA damage, and this strategy is selectively employed for the rapid


G C T U A T C C

C G A G T A G G

G C T A T C C

C G A G T A G G

G C T A T C C

C G A G T A G G

UURACIL DNAGLYCOSYLASE

AP ENDONUCLEASE AND PHOSPHODIESTERASEREMOVE SUGAR PHOSPHATE

DNA helixwith missingbase

DNA POLYMERASE ADDS NEWNUCLEOTIDE, DNA LIGASESEALS NICK

MBoC6 m5.48/5.42

deaminated C

DNA helixwith single-nucleotide gap



pyrimidine dimer



C T A

C G G T C T A C T A T G

G

5'

3'

5'

3'

EXCISIONNUCLEASE

DNAHELICASE

DNA POLYMERASEPLUS DNA LIGASE

DNA helixwith 12-nucleotide gap

(A) BASE EXCISION REPAIR (B) NUCLEOTIDE EXCISION REPAIR





G C T C A T C C

C G A G T A G G

Figure 5– 41 A comparison of two major DNA repair pathways. (A) Base excision repair. This pathway starts with a DNA glycosylase. Here, the enzyme uracil DNA glycosylase removes an accidentally deaminated cytosine in DNA. After the action of this glycosylase (or another DNA glycosylase that recognizes a different kind of damage), the sugar phosphate with the missing base is cut out by the sequential action of AP endonuclease and a phosphodiesterase. (These same enzymes begin the repair of depurinated sites directly.) The gap of a single nucleotide is then filled by DNA polymerase and DNA ligase. The net result is that the U that was created by accidental deamination is restored to a C. AP endonuclease is so-named because it recognizes any site in the DNA helix that contains a deoxyribose sugar with a missing base; such sites can arise either by the loss of a purine (apurinic sites) or by the loss of a pyrimidine (apyrimidinic sites). (B) Nucleotide excision repair. In bacteria, after a multienzyme complex has recognized a lesion such as a pyrimidine dimer (see Figure 5–39), one cut is made on each side of the lesion, and an associated DNA helicase then removes the entire portion of the damaged strand. The excision repair machinery in bacteria leaves the gap of 12 nucleotides shown. In humans, once the damaged DNA is recognized, a helicase is recruited to unwind the DNA duplex locally. Next, the excision nuclease enters and cleaves on either side of the damage, leaving a gap of about 30 nucleotides. The nucleotide excision repair machinery in both bacteria and humans can recognize and repair many different types of DNA damage.

2.2. Réparation des lésions d’un brin 2.2.3. Système BER (Base Excision Repair)


- hydrolyse de la liaison glycosidique (entre base et sucre) par ADN glycosylase

- création site AP (abasique)

- clivage liaison phosphodiester par AP endonucléase → libération unité sucre-phosphate

- remplissage de la brèche par ADN polymérase

- fermeture de la brèche par ADN ligase


36

2.3. Réparation des lésions simultanées des deux brins


- induites par radiations ionisantes ou stress cellulaire (agents oxydants, produits métaboliques)

- situation critique : 2 brins atteints, information perdue sur un court fragment d’ADN

- dégradation des chromosomes si absence de réparation

- ATM impliquée dans signalisation des cassures double brins, production d’enzymes de réparation, arrêt du cycle cellulaire

25/06/2014

35

69

(Adapté de Human Mol Gen, 2006)

b) Aspect cellulaire

III. Les Phases du cycle 3. G2

70

c) Réponse aux lésions de l’ADN

(ATM : Ataxia-telangiectasia-mutated)

III. Les Phases du cycle 3. G2

cf checkpoint vérification de l’ADN avant entrée en phase M


37

2.3. Réparation des lésions simultanées des deux brins


- 2 mécanismes : - recombinaison homologue (Homologous Recombinaison HR) - jonction non homologue des extrémités (Non-Homologous End Joining NHEJ)

- souvent avant réplication de l’ADN - assez simple - perte de séquence

- souvent après réplication de l’ADN, avant mitose - plus complexe - pas de perte de séquence

275

types of aberrant chromosomes are missegregated during cell division. As previ-ously discussed, the specialized structure of telomeres prevents the natural ends of chromosomes from being mistaken for broken DNA and “repaired” in this way.

A much more accurate type of double-strand break repair occurs in newly rep-licated DNA (Figure 5–45B). Here, the DNA is repaired using the sister chromatid as a template. This reaction is an example of homologous recombination, and we consider its mechanism later in this chapter. Most organisms employ both non-homologous end joining and homologous recombination to repair double-strand breaks in DNA. Nonhomologous end joining predominates in humans; homol-ogous recombination is used only during and shortly after DNA replication (in S and G2 phases), when sister chromatids are available to serve as templates.

DNA REPAIR

Figure 5–45 Two ways to repair double-strand breaks. (A) Nonhomologous end joining alters the original DNA sequence when repairing a broken chromosome. The initial degradation of the broken DNA ends is important because the nucleotides at the site of the initial break are often damaged and cannot be ligated. Nonhomologous end joining usually takes place when cells have not yet duplicated their DNA. (B) Repairing double-strand breaks by homologous recombination is more difficult to accomplish but restores the original DNA sequence. It typically takes place after the DNA has been duplicated (when a duplex template is available) but before the cell has divided. Details of the homologous recombination pathway are presented in the following section (see Figure 5–48).

Figure 5–46 Nonhomologous end joining. (A) A central role is played by the Ku protein, a heterodimer that grasps the broken chromosome ends. The additional proteins shown are needed to hold the broken ends together while they are processed and eventually joined covalently. (B) Three-dimensional structure of a Ku heterodimer bound to the end of a duplex DNA fragment. The Ku protein is also essential for V(D)J joining, a specific recombination process through which antibody and T cell receptor diversity is generated in developing B and T cells (discussed in Chapter 24). V(D)J joining and nonhomologous end joining show many similarities in mechanism but the former relies on specific double-strand breaks produced deliberately by the cell. (B, from J.R. Walker, R.A. Corpina, and J. Goldberg, Nature 412:607–614, 2001. With permission from Macmillan Publishers Ltd.)

processing ofDNA ends processing of 5′ ends

by nuclease

end joining homologousrecombination

damage repaired accurately using sister chromatid as the template

deletion of DNA sequence

double-strand break

(A) NONHOMOLOGOUS END JOINING (B) HOMOLOGOUS RECOMBINATION

sisterchromatids

MBoC6 m5.51/5.46

5′3′

5′3′

double-strand break in DNA

MBoC6 m5.52/5.47

END RECOGNITION BYKu HETERODIMERS

ADDITIONAL PROTEINS

PROCESSING OF DNA ENDS

LIMITED REPAIR SYNTHESIS

LIGATION

repaired DNA has generally suffered a deletionof nucleotides

(A)

(B)


38

2.3. Réparation des lésions simultanées des deux brins 2.3.1. Jonction non homologue des extrémités


- réparation de la brèche par simple jonction des extrémités - pas de restitution de la séquence parentale - pourtant assez fréquente chez eucaryotes supérieurs : risque minime de changement

de phénotype (% ADN codant des protéines très faible) - potentiellement fortement mutagène

275

types of aberrant chromosomes are missegregated during cell division. As previ-ously discussed, the specialized structure of telomeres prevents the natural ends of chromosomes from being mistaken for broken DNA and “repaired” in this way.

A much more accurate type of double-strand break repair occurs in newly rep-licated DNA (Figure 5–45B). Here, the DNA is repaired using the sister chromatid as a template. This reaction is an example of homologous recombination, and we consider its mechanism later in this chapter. Most organisms employ both non-homologous end joining and homologous recombination to repair double-strand breaks in DNA. Nonhomologous end joining predominates in humans; homol-ogous recombination is used only during and shortly after DNA replication (in S and G2 phases), when sister chromatids are available to serve as templates.

DNA REPAIR

Figure 5–45 Two ways to repair double-strand breaks. (A) Nonhomologous end joining alters the original DNA sequence when repairing a broken chromosome. The initial degradation of the broken DNA ends is important because the nucleotides at the site of the initial break are often damaged and cannot be ligated. Nonhomologous end joining usually takes place when cells have not yet duplicated their DNA. (B) Repairing double-strand breaks by homologous recombination is more difficult to accomplish but restores the original DNA sequence. It typically takes place after the DNA has been duplicated (when a duplex template is available) but before the cell has divided. Details of the homologous recombination pathway are presented in the following section (see Figure 5–48).

Figure 5–46 Nonhomologous end joining. (A) A central role is played by the Ku protein, a heterodimer that grasps the broken chromosome ends. The additional proteins shown are needed to hold the broken ends together while they are processed and eventually joined covalently. (B) Three-dimensional structure of a Ku heterodimer bound to the end of a duplex DNA fragment. The Ku protein is also essential for V(D)J joining, a specific recombination process through which antibody and T cell receptor diversity is generated in developing B and T cells (discussed in Chapter 24). V(D)J joining and nonhomologous end joining show many similarities in mechanism but the former relies on specific double-strand breaks produced deliberately by the cell. (B, from J.R. Walker, R.A. Corpina, and J. Goldberg, Nature 412:607–614, 2001. With permission from Macmillan Publishers Ltd.)

processing ofDNA ends processing of 5′ ends

by nuclease

end joining homologousrecombination

damage repaired accurately using sister chromatid as the template

deletion of DNA sequence

double-strand break

(A) NONHOMOLOGOUS END JOINING (B) HOMOLOGOUS RECOMBINATION

sisterchromatids

MBoC6 m5.51/5.46

5′3′

5′3′

double-strand break in DNA

MBoC6 m5.52/5.47

END RECOGNITION BYKu HETERODIMERS

ADDITIONAL PROTEINS

PROCESSING OF DNA ENDS

LIMITED REPAIR SYNTHESIS

LIGATION

repaired DNA has generally suffered a deletionof nucleotides

(A)

(B)

- reconnaissance des extrémités par protéines Ku (hétérodimères) + autres protéines

- jonction des extrémités → perte de certains nucléotides


39

2.3. Réparation des lésions simultanées des deux brins 2 .3.2. Système de recombinaison

homologue (HR)

2. MÉTHODES DE RÉPARATION 279

DNA polymerase utilizes a pristine template to restore damaged DNA. However, instead of using the partner complementary strand as a template, as occurs in most DNA repair pathways, homologous recombination exploits a complemen-tary strand from a separate DNA duplex.

Strand Exchange Is Carried Out by the RecA/Rad51 ProteinOf all the steps of homologous recombination, strand exchange is the most diffi-cult to imagine. How does the invading single strand rapidly sample a DNA duplex for homology? Once the homology is found, how does the exchange occur? How is the inherent stability of the template double helix overcome?

The answers to these questions came from biochemical and structural stud-ies of the protein that carries out these feats, called RecA in E. coli and Rad51 in virtually all eukaryotic organisms. To catalyze strand exchange, RecA first binds cooperatively to the invading single strand, forming a protein–DNA filament that forces the DNA into an unusual configuration: groups of three consecutive nucleotides are held as though they were in a conventional DNA double helix but, between adjacent triplets, the DNA backbone is untwisted and stretched out (Figure 5–49). This unusual protein–DNA filament then binds to duplex DNA

HOMOLOGOUS RECOMBINATION

daughterduplex DNAmolecules

DOUBLE-STRAND BREAK IS ACCURATELY REPAIRED

double-strand break

MBoC6 m5.59/5.49

5′3′

3′5′

5′5′

5′

5′

5′

5′3′

3′3′

3′5′

5′5′3′

3′5′

5′3′

3′5′

5′3′

3′5′

5′3′

3′5′

5′

5′

3′

5′3′

3′5′

NUCLEASE DIGESTS 5′ ENDSOF BROKEN STRANDS

STRAND EXCHANGE BYCOMPLEMENTARY BASE-PAIRING

REPAIR POLYMERASE SYNTHESIZES DNA (GREEN)USING UNDAMAGED DNA AS TEMPLATE

INVADING STRAND RELEASED; BROKENDOUBLE HELIX RE-FORMED

DNA LIGATION

DNA SYNTHESIS CONTINUES USING STRANDSFROM DAMAGED DNA AS TEMPLATE

Figure 5–48 Mechanism of double-strand break repair by homologous recombination. This is the preferred method for repairing DNA double-strand breaks that arise shortly after the DNA has been replicated, while the daughter DNA molecules are still held close together. In general, homologous recombination can be regarded as a flexible series of reactions, with the exact pathway differing from one case to the next. For example, the length of the repair “patch” can vary considerably depending on the extent of 5ʹ processing and new DNA synthesis, indicated in green.

- méthode de choix pour réparer les cassures double brins, après la réplication lorsque les molécules d’ADN sont encore proches → très efficace

- séries de réactions, différentes selon les cas

- utilisation des informations portées par chromosome homologue intact (chaque chromosome porte une copie de l’information)

- création d’une brèche, mais réparation sans perte d’information

- protéines spécialisées : - recombinases Rec A, B, C chez procaryotes - Rad51 chez eucaryotes


40

2.3. Réparation des lésions simultanées des deux brins 2.3.2. Système de recombinaison homologue (HR)


Pour info :284 Chapter 5: DNA Replication, Repair, and Recombination

Figure 5–55 A Holliday junction. The initially formed structure (A) is usually drawn with two strands crossing, as in Figure 5–54. An isomerization of the Holliday junction (B) produces an open, symmetrical structure that is bound by specialized proteins. (C) These proteins “move” the Holliday junctions by a coordinated set of branch-migration reactions (see Figure 5–57 and Movie 5.8). (D) Structure of the Holliday junction in the open form depicted in (B). The Holliday junction is named for the scientist who first proposed its formation. (PDB code: 1DCW.)

5′

5′

3′

3′5′

5′

5′ 3′

3′

3′

direction of branch migration

MBoC6 m5.58/5.57

branch point

ATP

ADP

ATP

ADP

Figure 5–56 Simplified view of branch migration. In branch migration, base pairs are continually broken and formed as the branch point moves. Although branch migration can happen spontaneously on naked DNA molecules, the process is inefficient and the branch moves back and forth at random. In the cell, branch migration is carried out using specialized proteins and ATP hydrolysis to ensure that, as shown, the branch moves rapidly and in one direction. As shown in Figure 5–57, branch migrations often occur at Holliday junctions, where two branch-migration reactions are coupled.

(D)

openform

MBoC6 m5.61/5.56

branchmigration

(C)(B)(A)

DNA (see Figure 5–21). A specialized nuclease then rapidly degrades the ends bound by Spo11, removing the protein along with the DNA and leaving protrud-ing 3ʹ single-strand ends.

At this point, many of the recombination reactions resemble those described above for the repair of double-strand breaks; indeed, some of the same proteins are used for both processes. However, several meiosis-specific proteins direct them to perform their tasks somewhat differently, resulting in the distinctive outcomes observed for meiosis. Another important difference is that, in meiosis, recombi-nation occurs preferentially between maternal and paternal chromosomal homo-logs rather than between the newly replicated, identical DNA duplexes that pair in double-strand break repair. In the sections that follow, we describe in more detail those aspects of homologous recombination that are especially important for meiosis.

Holliday Junctions Are Formed During MeiosisOf special importance in meiosis is an intermediate known as a Holliday junc-tion or cross-strand exchange (Figure 5–55). Each Holliday junction can adopt multiple conformations and a special set of recombination proteins binds to, and thereby stabilizes, the open, symmetric isomer.

Specialized proteins that bind to Holliday junctions can catalyze a reaction known as branch migration (Figure 5–56), whereby DNA is spooled through the Holliday junction by continually breaking and re-forming base pairs (Figure 5–57). In this way, the Holliday junction proteins use ATP hydrolysis to expand the region of heteroduplex DNA initially created by the strand-exchange reaction. In meiosis, heteroduplex regions often “migrate” thousands of nucleotides from the original site of the double-strand break. As shown in Figure 5–54, Holliday junc-tions usually occur in pairs, known as double Holliday junctions.

Homologous Recombination Produces Both Crossovers and Non-Crossovers During MeiosisAs shown in Figure 5–54, there are two basic outcomes of homologous recom-bination during meiosis. In humans, approximately 90% of the double-strand breaks produced during meiosis are resolved as non-crossovers (see right side of Figure 5–54). Here, the two original DNA duplexes separate from each other in a form unaltered except for a region of heteroduplex that formed near the site of the original double-strand break. This set of reactions resembles that described above for the repair of double-strand breaks (see Figure 5–48).

The other outcome is more profound: a double Holliday junction is formed and is cleaved by specialized enzymes to create a crossover (see left side of Figure 5–54). The two original portions of each chromosome upstream and downstream

formation de jonctions de Holliday


41

RÉSUMÉ DES PRINCIPAUX SYSTÈMES DE RÉPARATION


U X

X

X

X

X(CH3)n

A G

X

X

XX

AA T

T

incorporation d’uracile

modification de base

alkylation

adduits volumineux

pontage intra-brin

5’

5’

coupure double-brins

pontage inter-brin

site abasique

coupure simple-brin

dimère de thymine

mésappariement

Principaux types de lésions de l’ADN

BER

NER

HR/NHEJ

MMR

Base Excision Repair

Nucleotide Excision Repair

MisMatch Repair

Homologous Recombination Non Homologous End Joining

ADN réparation (8) Méthodes de réparation (2)

Réparation directe du dommage

Même type de réaction pour les groupements ethyl Chez l’‛homme, les mutations de MGMT sont associées à une fréquence accrue de cancer (rôle suppresseur de tumeur)

Alkylation

methylguanine methyltransferase

MGMT

Réparation des méthylations de guanine

Réparation des dimères de pyrimidine

Action d’‛une photolyase

DNA natif

DNA avec

dimère

liaison de la photolyase

au dimère

Absorption de lumière (>300 nM)

Réversion et correction de

l’erreur largage de la lyase

Correction immédiate3. correction immédiate

erreurs d’incorporations§1/104 pb vs 1/108 pb final

arrêt de la polyméraseactivité 3’-5’ exonucléase reprise de synthèse 5’- 3’

photolyases

réversion directe par des alkyltransférases (ex: O6-méthylguanine- DNA méthyl transférase = MGMT)

activité 3’-5’ exonucléase des polymérases

ADN avec dimère de T liaison de la photolyase au dimère

absorption de la lumière réversion et correction de l’erreur, largage de la lyase


42

SOMMAIRE









43

3.1. Principe

3. SYNTHÈSE TRANSLÉSIONNELLE

- ≠ système de réparation mais TOLÉRANCE

- aide la cellule à survivre en tolérant certains dommages de l’ADN

- objectif : éviter le blocage de la réplication, lorsque la lésion empêche la progression de la fourche de réplication

→ remplacement de l’ADN polymérase réplicative par une ADN polymérase translésionnelle (“by-pass polymérase) :

- différents types selon lésion - efficacité et FIDÉLITÉS VARIABLES

→ insertion de quelques nucléotides jusqu’à une position où la matrice devient à nouveau acceptable pour la polymérase réplicative


44


repair. Ionizing radiation, replication errors, oxidizing agents, and other metabo-lites produced in the cell cause breaks of this type. If these lesions were left unre-paired, they would quickly lead to the breakdown of chromosomes into smaller fragments and to loss of genes when the cell divides. However, two distinct mecha-nisms have evolved to deal with this type of damage (Figure 5–45). The simplest to understand is nonhomologous end joining, in which the broken ends are simply brought together and rejoined by DNA ligation, generally with the loss of nucleo-tides at the site of joining (Figure 5–46). This end-joining mechanism, which can be seen as a “quick and dirty” solution to the repair of double-strand breaks, is common in mammalian somatic cells. Although a change in the DNA sequence (a mutation) results at the site of breakage, so little of the mammalian genome is essential for life that this mechanism is apparently an acceptable solution to the problem of rejoining broken chromosomes. By the time a human reaches the age of 70, the typical somatic cell contains over 2000 such “scars,” distributed through-out its genome, representing places where DNA has been inaccurately repaired by nonhomologous end joining. But nonhomologous end joining presents another danger: because there seems to be no mechanism to ensure that two ends being joined were originally next to each other in the genome, nonhomologous end joining can occasionally generate rearrangements in which one broken chromo-some becomes covalently attached to another. This can result in chromosomes with two centromeres and chromosomes lacking centromeres altogether; both

covalent modificationsto sliding clamp whenpolymerase encountersDNA damage

5′3′

sliding clamp DNA damage

replicative DNApolymerase released

DNA synthesis

loading of translesion polymeraseby assembly factors

translesion DNApolymerase

removal of covalent modifications, reloading of replicativeDNA polymerase, DNA synthesis continues

MBoC6 n5.100/5.45

Figure 5–44 Translesion DNA polymerases can use damaged templates. According to this model, a replicative polymerase stalled at a site of DNA damage is recognized by the cell as needing rescue. Specialized enzymes covalently modify the sliding clamp (typically, it is ubiquitylated—see Figure 3–69) which releases the replicative DNA polymerase and, together with damaged DNA, attracts a translesion polymerase specific to that type of damage. Once the damaged DNA is bypassed, the covalent modification of the clamp is removed, the translesion polymerase dissociates, and the replicative polymerase is brought back into play.


3.1. Principe

- reconnaissance du dommage et arrêt de la polymérase réplicative

- modifications covalentes du clamp → relargage de la polymérase réplicative

- recrutement d’une polymérase translésionnelle spécifique du dommage

- synthèse d’ADN, ± fidèle à la matrice

- retrait des modifications covalentes du clamp

- dissociation de la polymérase translésionnelle, et retour de la polymérase réplicative


45

3.2. Système SOS procaryote


- ensemble de gènes (environ 30) impliqué dans la réplication, la réparation de l’ADN et la division cellulaire

- contrôle de l’expression de ces gènes par altération de l’ADN

si arrêt de la réplication :

→ recrutement SSB et recombinase RecA

→ activation activité protéolytique de RecA

→ clivage LexA : régresseur de l’expression des gènes codant des protéines de réparation (opéron lexA)

→ clivage par RecA permet donc l’expression de ces gènes, notamment polymérase translés ionnel le qu i incorpore de préférence des adénines

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46

SOMMAIRE









47

4. PATHOLOGIES ASSOCIÉES

- tout au long de la séquence : possibilité de mutations muettes, sauf - codon initiateur ATG, qui code méthionine - codon TGG du tryptophane → apparition d’un codon stop prématuré

- chez organismes diploïdes : chaque gène présent en 2 exemplaires (2 allèles) si mutation sur 1 seul des allèles : - mutation récessive : l’effet est masqué par l’allèle sauvage qui remplit la fonction

normale → phénotype uniquement chez individus homozygotes (2 parents hétérozygotes pour la

mutation) - mutation dominante : l’effet se manifeste malgré la présence de l’allèle sauvage → phénotype chez individus hétérozygotes

- chez organismes haploïdes : toutes les mutations peuvent introduire un phénotype → levures : modèles qui permettent d’obtenir facilement des mutations


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Une mutation peut entraîner :

- une perte de fonction : - le gène ne s’exprime plus - le gène s’exprime mais la protéine mutée ne peut plus remplir sa fonction → mutation perte de fonction récessive → mais parfois, nécessite de l’expression des 2 allèles pour quantité protéique suffisante à la fonction → mutation perte de fonction dominante

- un gain de fonction : - le gène s’exprime plus fortement - la protéine mutée peut avoir :

- une fonction plus intense - une fonction qui devient constitutive alors qu’elle était indicible - une nouvelle fonction → mutation gain de fonction peuvent être dominantes


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Mutations peuvent être à l’origine :

- de maladies monogéniques : touchent un gène unique (drépanocytose, mucoviscidose) → souvent héréditaires

- de maladies multigéniques : touchent plusieurs gènes différents → souvent acquises (cellules somatiques)

Défauts dans les systèmes de réparation de l’ADN : à l’origine de nombreux cancers

- Xeroderma Pigmentosum (ou “maladie des enfants de la lune”) : cancers de la peau, provoqués par dysfonctionnement du système NER (mutation des gènes XP)

- cancer du colon non polyposique (polype = croissance anormale de tissus sur des muqueuses) : dysfonctionnement du système MMR

- syndrome de Bloom : dysfonctionnement du système de réparation des cassures (mutations de l’hélicase) → forte sensibilité aux rayons X et au soleil

- certaines formes familiales de cancer du sein : mutations dans les gènes BRCA1 et BRCA2 (codent protéines accessoires à la recombinase Rad51, système de recombinaison homologue)

- etc… ☹

Documents

Chapitre 4 Réparation de l’ADNintranet.supbiotech.fr/courses/BIOLOGIE/document/2016-2017/Cours/... · → sauvegarde de l’information génétique -modifications de l’ADN car