Évolution des génomes de bactériophages

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Évolution des génomes de bactériophages

Jihane Amarir-Bouhram

To cite this version:

Jihane Amarir-Bouhram. Évolution des génomes de bactériophages. Sciences agricoles. Université Paris Sud - Paris XI, 2012. Français. �NNT : 2012PA112036�. �tel-00923139�

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Table des matières

Liste des abréviations ... 6

Liste des Figures ... 8

Liste des Tables ... 10

INTRODUCTION ... 11

Chapitre I. La recombinaison homologue chez les bactéries. ... 14

I. Rôle et structure de la recombinase RecA. ... 15

II. La recombinaison homologue chez

... 15

1. L’étape pré-synaptique. ... 16

2. L’étape synaptique. ... 18

3. L’étape post-synaptique. ... 19

III. La recombinaison homologue indépendante de RecBCD. ... 19

IV. La recombinaison homologue entre séquences d’ADN non identiques chez

... 20

Chapitre II. La recombinaison homologue chez les bactériophages. ... 22

I. La recombinase UvsX du bactériophage T4. ... 22

II. La recombinaison homologue chez le bactériophage Lambda. ... 23

1. Rôle et structure de la recombinase Red

... 24

2.

... 25

,,, #0*11*-2$3$4+&.0567&%2318$&9-24&1*-&/$.!"

... 26

1.

L’appariement d’ADN simple brin

... 27

2.

L’appariement d’ADN simple brin

... 27

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? ... 30

I. L’invasion de brin

. ... 30

II. L’invasion de brin

... 31

Chapitre IV

-elle un cofacteur de RecA? ... 37

I. Rad52 chez les eucaryotes ... 37

II. Les recombinases phagiques de la superfamille Rad52 ayant une activité cofacteur de RecA. 39 1. Red

est un cofacteur de RecA lorsque Lambda ne peut pas se répliquer ... 39

2. Le recombineering Red

-dépendant est diminué dans un mutant

... 40

3.

Sak et Sak3, deux recombinases de bactériophages infectant des bactéries lactiques, sont des cofacteurs de RecA

... 41

Chapitre V : Synthèse, formulation de la question poursuivie lors de la thèse ... 43

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MATERIEL ET METHODES ... 44

I. Souches et plasmides ... 44

1. Souches ... 44

2. Plasmides ... 44

II. Préparation des cellules électro-compétentes ... 45

,,,

Le système expérimental d’appariement d’ADN simple brin

... 46

1. Description. ... 46

2. La quantité d’oligonucléotide. ... 46

3. La taille de l’oligonucléotide. ... 47

4. Meilleure efficacité de recombinaison avec un oligonucléotide complémentaire au brin tardif. ... 47

5. Marquages ... 48

1. L’expression des recombinases phagiques testées par Western Blot. ... 48

2. L’expression des recombinases phagiques testées par marquage radioactif ... 49

6. Expérience de complémentation de la sensibilité aux UV de la souche AB1157

. ... 50

RESULTATS ... 51

Chapitre I ... 51

Résumé ... 51

Article ... 53

Chapitre II ... 97

Introduction ... 97

,

Activité résiduelle d’appariement d’ADN simple brin chez

. ... 99

II. Les recombinases RecA et UvsX se comportent différemment pour l’appariement d’ADN simple brin

. ... 100

,,,

Le domaine ATPase d’UvsX est essentiel à l’appariement d’ADN simple brin

contrairement à celui de Sak4. ... 101

,9

Le domaine C-ter d’UvsX est impliqué dans le mécanisme d’appariement simple brin. .... 102

9 L’activité de la recombinase Sak4 est inactivée par la mutation K123A

... 103

9,

La recombinase Sak4 du bactériophage HK620 apparie efficacement l’ADN simple brin en présence de ses cofacteurs. ... 104

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. 105

Gam stimule l’activité d’appariement d’ADN simple brin de Red

... 108

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DISCUSSION ... 110

I.

possède une activité d’appariement d’ADN simple brin en contexte

... 110

II. RecA ne fait pas d’appariement d’ADN simple brin

... 111

III. UvsX fait de l’appariement simple brin

... 112

IV. L’activité d’appariement d’ADN simple brin

de Sak4 est indépendante de son site de fixation de l’ATP ... 112

V. Les recombinases de bactériophage font-elles toutes de l’appariement d’ADN simple brin lorsque les séquences divergent ? ... 113

VI. Gam favorise l’appariement simple brin

... 116

Conclusion générale ... 118

BIBLIOGRAPHIE ... 119

Annexe ... 124

Résumé : ... 134

Summary : ... 134

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