procaryotes : a single circular chromosome typically 5.106 base pairs

eucaryotes : several linear chromosomestypically 3.109 base pairs

22 autosomal chromosome

pairs

2 sexual chromosomes

Example : human genome

+ about hundred circular

mitochondrial DNA molecules

telomere

centromere

DNA is organized in chromosomes

1

5 µm

Structure and localization of chromosomes

Chromosomes are made of DNA and proteins (chromatin). Histones make DNA more compact and regulate its accessibility (nucleosomes). Transcription factors control gene expression. Replication factors catalyze DNA replication during the S phase of the cell cycle. Individual chromosomes can be observed during mitosis, one step of cell division (chromatin condensation). Centromeres are contact points between pairs of chromosomes. Telomeres are chromosome ends. A diploid (haploid) cell possesses 2 (1) set(s) of chromosomes

2

Regulation of DNA accessibility play a crucial role during transcription and replication

From ENCODE, an encyclopedy of DNA elements : http://encodeproject.org/ENCODE/ 3

DNA replication

DNA replication is semi-conservative

DNA polymerases

Replication origins

Assembly of the replication fork

Further readings : http://www.dnaftb.org/dnaftb/

http://www.dnareplication.net/

1 ADN 2 ADN

4

DNA replication is semi-conservative

M. Meselson & P Stahl Proc. Nat. Ac. Sci. 1958

5

6

The cell cycle

Gap 1

DNA Synthesis

Mitosis

Gap 2

In the resting state (G0), cells do not divide

G0

6

Flow cytometry

Up to 8 fluorophores can be simultaneously analyzed

http://www.abcam.com/

Fluorescence level

Forward and side scattering is used to analyze cell size and granularity

Analysis rate ≈ 10000 cells/secSeveral analysis in parallel

7

The Fluorescence Activated Cell Sorter (FACS)

8

9

DNA synthesis is catalyzed by DNA-dependent DNA polymerases

dNTP template strand

strand to be synthesized

DNA polymerization takes place in the 5’ to 3’ direction DNA polymerase requires a template and a primer

dATPdTTP

dCTPdGTP

GGATCCTTAGAACCTTGGCCCGGGCCTAGGAATCTTGGAACCGGGCCC

DNA polymerase nucleotides

GGATCCCTAGGAATCTTGGAACCGGGCCC

template

primer5’ 5’

PPiPPi

PPi

PPiPPi

PPi

Stryer et al. Biochemistry, Freeman Edt9

DNA replication is catalyzed by a DNA-dependant DNA polymerase in the 5 ’ to 3 ’ direction starting at double strand DNA or at a DNA-RNA hybrid A primase synthesize a RNA primer to initiate replication DNA polymerases are processive : processivity is the number of phosphodiester bonds that a single enzyme is able to catalyze before dissocation

DNA replication requires a primase to start

dNTP

template strand

strand to be synthesized

10

11

Okazaki fragments

RNA primase

Leading and lagging strands

Size of Okasaki fragments : eukaryotes 200 bp

Alberts et al. MBOC, Garland Edt11

12

5’3’

dNTPRNA primer

5’3’

NTPprimase

Replication fork

DNAPol

DNA helicase

DNA helicase

On the « leading strand », DNA is continuously synthesized

12

13

5’ 3’

RNA primer

dNTP RNA primer

5’ 3’

NTP

DNAPol primase

5’ 3’

dNTP RNA primer

ligase

5’ 3’

RNA primer

RNAse and DNAPol

Replication fork

DNAPol

DNA helicase

DNA helicase

DNA helicase

DNA helicase

On the « lagging strand », DNA is synthesized discontinuously

13

14

The core of the eukaryote replication complex

Movies 5.1 (Molecules and Complexes) and 5.4 (Cell functions) Mol. Biol. Cell

Linda B. Bloom, University of Floridahttp://www.med.ufl.edu/IDP/BMB/bmbfacultypages/lindabloom.html

Eukaryote cells possesses several DNA polymerases (> 15) nucleus 250 kDa DNA primase, lagging strand nucleus 170 kDa leading strand

nucleus 260 kDa lagging strand, DNA repair

DNAPol

DNAPol

DNAPol primase

14

15

Main components of the DNA replication complex

DNA polymerase – primase primer RNA synthesisDNA polymerase DNA synthesis, leading+lagging strands

Replication protein C* load PCNA on DNAProliferating cell nuclear antigen (PCNA) sliding clamp ensuring processivity

Topoisomerase Adjusts DNA supercoilingHelicase* Unwinds DNA into strands

Replication protein A single strand DNA binding proteinFlap endonuclease 1 removes RNA 5’-flapDna2RNase H1 removes RNADNA ligase 1 joins Okasaki fragments

* uses ATP The replisome

The catalytic core

Maga and Hübscher 1996 Biochemistry 35: 5764-5777Waga and Stillman 1994 Nature 269: 207-212Frouin et al. 2003 EMBO reports 4: 666-670Hübscher and Yeon-Soo Seo 2001 Mol. Cells 12: 149-157

Cyclin A, cyclin B1Cyclin dependent kinase 1, 2 (CDK1, CDK2)

+ 11 other proteins…

Temporal regulation

15

16

The central role of PCNA

PCNA (proliferating cell nuclear antigen) is a homotrimeric protein that helps DNA polymerase processivity in eukaryotic cells. During the S-phase, it assembles around DNA and form a DNA clamp.

PCNA associates with RFC, DNA polymerases and , Fen1/Dna2, Lig1 (+ 15 other proteins !)

PCNA is also involved in DNA repair mechanisms

At 3’ OH end : RFC displaces Pol- and loads PCNA + Pol/At the flap structure :

RFA dissociates Pol from PCNAPCNA recruits Fen1/Dna2 which cleaves the flap

structurePCNA recruits Lig1 that joins the DNA fragments

PDB 1AXC

Maga and Hübscher 2003Journal of Cell Science 116: 3051-3060

16

17

Replication is coordinated at replication factories

Visualization of DNA replication in living cells using GFP-PCNA FRAP experiments shows that PCNA is stably associated to replication factories

Essert et al. 2005 Mol. Cell Biol. 25 : 9350-59

PCNA

GFP

17

18

Replication is coordinated at replication factories

Visualization of DNA replication in living cells using GFP-PCNA FRAP experiments shows that PCNA is stably associated to replication factories

Essert et al. 2005 Mol. Cell Biol. 25 : 9350-5918

19

There are about 100-1000 replication origins per chromosome Replication origins are recognized by specific protein complexes : ORC ‘origin recognition complex) and MCM (minichromosome maintenance complex) Replication speed : 10-50 bp/s The onset of DNA replication is triggered by « cell division cycle dependant kinases » (CDK)

Replication starts at replication originsORC : origin replication complexMCM : minichromosome maintenance complexReplisome

1. Activation

2. Extension

3. Termination

19

Les recombinaisons: modifications aléatoires et programmées du génome

ADN1 + ADN2 ADN3 + ADN4

Mécanisme moléculaire de la recombinaison homologue

La recombinaison de sites spécifiques

La conjugaison, mécanisme de la parasexualité bactérienne

La recombinaison VDJ, un des éléments de la diversité des anticorps

et des TCR

Le crossing-over durant la méiose accroît la diversité génomique de

la population

Les transposons et les virus, séquences d’ADN mobiles

20

homology

cleavage

ligation

exchange

displacement

Holliday junction

cleavage

ligation

The mechanism of homologuous recombination

21

ATP binding site

ATP hydrolysis

RecA proteins catalyze the exchange of DNA strands ...

Structure of a RecA polymer

22

… dans un seul sens

without RecA with RecA

Driving force : ATP hydrolysis

… in the 5’ to 3’ direction

23

Recombination events in cells

Example Cells Effect Effector proteins

Crossing-over Meiotic cells genome RecA-D like ( germinal cells) rearrangements proteins

Virus integration Host cell genome dormancy Integraselytic/lysogenic Integration Host phases Factor

Conjugation Bacteria gene exchange Integrase

VDJ recombination lymphocytes antibody and Rag1-2 TCR diversity

Transposons all cells genome Transposasesrearrangements

24

The two states of the bacteriophage Reversible recombination

DNA of the bacteriophage

DNA of E. coli

attP

attB

Recombinant DNA

IntegraseIntegration Host Factor

ExcisionaseIntegraseIntegration Host Factor

Example 1 : site-specific recombination of a virus

25

Integrase mechanism

phage DNA

E. Coli DNA

attP

attB

recombinant DNA

pairing, double cleavage, double exchange, ligation

26

Conformation 1 : phage and bacterial DNA separatedConformation 2 : phage and bacterial DNA fused

attB attP

bacterial DNA

phage DNA

27

Biswas et al. (2005) A structural basis for allosteric control of DNA recombination by λ integrase Nature 435 : 1059-1066

integration

excision

Phage integration in bacterial genome

28

Conjugation Reversible recombination

« female »

« male »

DNA

episome F

factor F

chromosome

Hfr chromosome

plasmide F ’

integration

excision

F’ plasmids often carry virulence factors

The F-factor allows gene exchange between bacteria

29

ampicillineR

blasticidineR

gène cible blasticidineR

Recombinaison (double

crossing-over)

WT

PHG1A

phg1a phg1bphg1a/b

PHG1B

Anti-PHG1B

Anti-PHG1A

Benzhegal et al. 2002

Applications de la recombinaison : invalidation de gène par insertion

30

Applications of recombination : the Cre-Lox system

Cre recombinase : a P1 phage enzyme that catalyzes recombination between two LoxP sequences :LoxP : ATAACTTCGTATAGCATACATTATACGAAGTTAT

Example : RIP-CreER transgenic mice have a tamoxifen inducible Cre-mediated recombination system driven by the rat insulin 2, Ins2, promoter. The transgene insert contains a fusion product involving Cre recombinase and a mutant form of the mouse estrogen receptor ligand binding domain. The mutant mouse estrogen receptor does not bind natural ligand at physiological concentrations but will bind the synthetic ligand, 4-hydroxytamoxifen. Restricted to the cytoplasm, the Cre/Esr1 protein can only gain access to the nuclear compartment after exposure to tamoxifen. When crossed with a strain containing a loxP site flanked sequence of interest, the offspring are useful for generating tamoxifen-induced, Cre-mediated targeted deletions. Tamoxifen administration induces Cre recombination in islet cells of the pancreas. About 100 loxP-flanked genes bearing strains are available at Jackson 31

Light chain of antibodies

Example 2 : genetic rearrangements in B lymphocytes

recombination

RAG : recombination activating genesRSS : recombination signal sequences

splicing

32

Transposons are mobile DNA sequences in genomes

excision insertiontranscriptiontraduction

transposase

example : Tn5 transposon and transposase 33

no specific insertion sites frequency of mobility: 10-6 per generation Abundance variable in genomes (10% in drosophila, 40% in men)

coat proteins use receptors to enter the cells

type I transposons (retrotransposons)

type II transposons

ARNm

resolvasetranscriptase réverse

transcriptase réverse

resolvase

cDNA

ARNm

transposase

activité derestriction activité

d'intégration

ADN excisé

DNA viruses

RNA viruses

Viruses and transposonsTransposons Viruses

34

entrée du viruspar fusion avecla membrane plasmiquegràce à des récepteursde la surface cellulaire

Pour les virus à ARNcopie en ADN par unetranscriptase réverseviraleContrôle de la cellule Intégration dans le génome

Silence expressionDormance

Productions de protéines etacides nucléiques virauxpar la cellule et enpaquetagede nouveaux virusDestruction de la cellule

Fast viruses

Slow viruses

Fast and slow viruses

35

The presence of transposons allows gene duplication, inversion or excision by homologous recombination

DELETION INVERSION

DUPLICATION

36

example of a diploid organism with 2 pairs of homologous chromosomes

MITOSIS

MEIOSIS

FECUNDATION

diploid

4 haploids

gametes

2 diploids

diploid

diploid

2 haploids

Mitosis, meiosis and fecundation

37

DNA replication

decondensation of chromosomes

separation of daughter cells (cytokinesis)

Chromosome condensationcentromere

s

Sister chromatides

separation of sister chromatides

Mitotic spindle

Mitosis : 1 diploid -> 2 diploids

38

DNA replication

separation of homologous

chromosomes

gametes

Chromosome condensationcentromere

Sister chromatids

Pairing of homologous

chromosomes

synaptolemalcomplex

1st mitosis

2nd mitosis

Meiosis : 1 diploid -> 4 haploids

39

« Crossing over »

Transmission de l ’ADN mitochondial

transmission presque exclusivement par la mère

simple

double

séquences homologuesfréquence : 1/107 paires de bases

chromosome paternel

chromosome maternel

Epigénétique

Certains gènes sont inactivés par méthylation, l ’état de méthylation peut être transmis aux cellules filles Exemple : inactivation d ’un des chromosomes X chez les femmes

Transmission non-mendélienne

40

réplication de l ’ADN

ségrégation des chromosomes homologues

gamètes

condensation des chromosomescentromère

chromatides sœurs

appariement des chromatides

homologues et crossing-over

Complexe synaptolemal

1ière mitose

2ième mitose

Recombinaison durant la méiose

41

Chez E. coli, la recombinaison homologue a lieu à des sites spécifiques appelés « chi site » dont la séquence est GCTGGTGG, situés environ toutes les 4000 paires de baseChez E. coli, la recombinaison est catalysée par l ’action de quatres protéines RecA, RecB, RecC et RecD

L’ADN simple brin est généré par l ’action d ’une hélicase et d’une endonuclease du complexe RecBCD

42

DNA repair

Molecular origin of DNA mutations

General repair mechanisms

The p53 protein controls DNA damage at a specific checkpoint

of the eukaryote cell cycle

43

Sources of DNA damage

Replication errors: DNA polymerase frequency 1/107

Molecular damages to DNA:

Origin DNA damage number/cell.day Possiblerepair

Exogenous sun (1h/day) T-T dimers 6-8.104 Ychemical adducts 102-105 N

(base modification)radioactivity single strand breaks 2-4.104 Y(natural double strand breaks ? ±background)

Endogenous temperature single strand breaks 2-4.104 Yfree radicals adducts/breaks 104 Ymetabolites adducts 102 Yviruses genome integration ? Ntransposons ? ? 44

DNA repair mechanisms

Damage type Repair

T-T dimers

Adducts

Single strand breaks

Double strand breaks

Restriction

Excision

Synthesis

Ligation

Excision

Recombination

Ligation

or direct ligation

Recognition

45

The COMET assay to measure DNA damages

also called single cell gel electrophoresis (SCGE)

46

Ames test (Salmonella-his reversion-test ) for mutagenicity

This experiment employed six strains of Salmonellatyphimurium histidine auxotroph mutants, deficient in the synthesis of histidine, an amino acid necessary for bacterial growth. The histidine auxotrophs will only grow in a medium containing sufficient histidine supplement. To revert to histidine production (prototrophy), or become his+,a reverse mutation must occur in the original his- mutation (found in one of the genes involving histidine biosynthesis). When plated onto an agar media containing a trace (1/1000 dilution) of histidine, only his+ revertants will grow to form a visible colony.

The presence of visible colonies signifies a reverse mutation. Each of the six bacterial strains carries a different type of mutation (Table 1), making it possible to assess the type of mutation caused by the chemical under examination. When a chemical mutagen is introduced into the bacterial population on a filter disc, a higher number of revertants will appear, signalling the chemical causes genetic mutations.

The Ames test includes using liver extract to simulate mammalian metabolic activity which may alter non-mutagenic chemicals to become mutagenic. The liver extract is generally obtained from rats treated with Aroclor 1254 to induce the presence of detoxifying enzymes.

Brian Krug: Ames Test: Chemicals to Cancer

Strain # S. typhimurium Type of Mutation Detected Strain Name1 TA98 detect frame-shift mutations2 TA100 detect base pair substitutions3 TA102 detect excision repair4 TA104 detect base-pair substitutions5 TA1534 detect frame-shift mutation6 TA1530 detect base pair substitutions

Inhibition zone

growth ring

chemical to be tested

47

Exemple of repair : thymine dimers

Tymine dimer repair enzyme : specific DNA endonuclease

(induced by UV light)

48

benzo[a]pyrene (BP)

Metabolism et carcinogenicity of Benzo[a]Pyrene

benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide

CYP1A1, CYP1A2epoxide hydrolase

the diol epoxide covalently binds to DNA (adduct)

Increased DNA

mutations & cancer

Benzo[a]pyrene is a product of incomplete combustion at temperatures between 300 and 600 °C. aromatic

molecule (L)

Aryl hydrocarbon

ReceptorAhR

AhR-L

induction of specific mRNA (AhRE)

AhR-L

GrowthDifferentiationMetabolism

(toxicity)

P450 cytochromes (phase I) : CYP1A1, CYP1A2, CYP1B1, CYP2S1

Phase II enzymes : GST, UGT(detoxification mechanism)

translocation to the nucleus

AhRE AhRE

49

Shimizu et al. (2000) PNAS 97 : 779-782Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor

Dossier INSERMDioxines dans l’environnement. Quels risques pour la santé ? http://ist.inserm.fr/basisrapports/rapport.html

Individual susceptibility to xenobiotics. Exemple of CYP genes

50

The rad genes in yeast

A systematic study was conducted in yeast to identify genes responsible for the cell sensitivity to radiation 55 “rad” genes were found. Most of these genes have counterparts in the human genome. From current estimates, 240 genes are involved in DNA repair in humans

51

P Perego (2000) Yeast Mutants As a Model System for Identification of Determinants of Chemosensitivity. Pharmacol Rev 52: 477–491

52

Cell cycle checkpoints

APOPTOSIS

p53

APOPTOSIS

Retinoblastoma protein (Rb)

APOPTOSIS

Anaphase Promoting Complex (APC)

Apoptosis is an organized (programmed) cell death mechanism

53

Apoptosis

Apoptosis is one form of programmed cell death, often observed in higher eukaryotes during development, selection of immune system cells, and cancer prevention by NKC

Apoptosis can be triggered by intracellular processes, such as DNA damages, or by extracellular molecules, for instance activation of the Fas receptor by the Fas ligand, or the secretion of permeabilizing molecules by NKC.

Apoptosis involves mitochondrial inactivation and the release of cytochrome c in the cytosol.

The lack of ATP induces phosphatidyl serine exposure to the plasma membrane (the “eat-me” signal) and cell blebbing.

Cell fragments are internalized by macrophages and digested. No inflammation (activation of the innate immune system) occurs .

See movie 18.1 apoptosis54

The p53 protein holds the cell cycle at the G1/S checkpoint in the presence of DNA damage

p53 is a tetrameric 393 aa protein

p53 consists of 3 domains :

1 100 200 300 393

transcription activation domain

DNA binding domain

regulatory domain

NLSphosphorylations

The transcription activation domain interacts with the Mdm2 protein that triggers p53 degradation.

The DNA binding domain interacts with a specific DNA sequence that controls p21CIP expression

The conformation and the localization of p53 is controlled by phosphorylation and acetylation

acetylations

p53 DNA binding domains in complex with DNA

55

TRANSCRIPTION of p21 inhibitor

Pcdk2

cyclineEp21

p53synthesis degradation

p53-PDNA polymerase

ADN intact damagedChk inactive activep53 absent bound to DNA

(mdm2) (phosphorylated)p21 repressed expressedCDK active inactiveCycle G1S G1 stop

p53-UbMdm2

+

Double strand break

Single strand break (30 to 40 bases lacking)

Base mispairing

Chk1/2 +

Mdm2 = murine double minute oncogeneChk = checkpoint kinasep21= CIP (cdk2 inhibiting protein) = WAF1 (Wild Type p53-activated fragment)

56

p53 mutations are found in 50% of human cancers

Arg248

Arg175, 249, 273, 282, Gly245

Mutation frequency

Séquence primaire de p53

These mutations decrease p53 interaction with DNA, which eliminates the G1/S restriction point controlled by the Cdk2-cyclinE complex 57

4. Natural Killer Cells and cancer prevention

Natural Killer Cells (NKC) are components of the (innate) immune systems. They are cytotoxic against tumor cells and cells infected by viruses. They also play an important role in graft rejection.

NKC are sensitive to the molecules present at the surface of the cells. All cells in the body express MHC-I complexes that present fragments of endogenous proteins synthesized in the cell. Any change in the nature of MHC-I or in the surface concentration of MHC-I leads to NKC activation

Upon activation, NKC bind to the target cell and locally release perforin and granzyme molecules at the plasma membrane of the target cell, which triggers apoptosis.

In addition, NKC are able to recognize and kill cells with antibodies bound at their surface (adaptative immune system). Antibodies directed against surface antigens are indeed often present in cancers.

Defects in NKC production severely increases the risk of cancer

Tumor cells develop inhibitors that prevent NKC activation

See movie 24.4 killer T cells58

proteins

DNA (desoxyribonucleic acid)

mRNA (ribonucleic acid)

Transcription factors

DNA state sensors

Repairenzymes

Replication factors

Apoptosis factors (mitochondrial inactivation, caspases)

p53

CELL DIVISION

CELL APOPTOSIS = CELL DEATH

UNCONTROLLED CELL DIVISION = CANCER

DNA repair mechanisms and cell fate

Radiations DNA damage apoptosisRapidly dividing cells are more sensitive to radiations 59

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Indirect carcenogenicity of dioxin

Dioxins occur as by-products in the manufacture of organochlorides, in the incineration of chlorine-containing substances such as PVC, in the bleaching of paper, and from natural sources such as volcanoes and forest fires.

Dioxins build up primarily in fatty tissues over time. The major source of dioxins is food, especially from animals. TCDD has a half-life of approximately 8 years in humans.

TCDD activates the AhR and thus induces CYP expression. This either increases or reduces carcinogenicity of other aromatic molecules such as Benzo[a]Pyrene and 7,12-dimethylbenz[a]anthracene, respectively.

Travailleurs exposés aux phénoxy-herbicides et aux chlorophénols. Exposition : 3 à 389 pg/g de matières grasses Teneur du lait maternel en France : 16,5 ± 5 pg/g de matières grasses Dossier INSERM Dioxines dans l’environnement.

60

22 paires de chromosomes autosomaux homologues Ci

p/Cim

2 chromosomes sexuels Xm/Yp

Père 22 paires de chromosomes autosomaux homologues Ci

p/Cim

2 chromosomes sexuels Xm/Xp

Mère

22 chromosomes autosomaux Ci

p ou Cim

1 chromosome sexuel Xm ou Yp

spermatozoïdes

22 chromosomes autosomaux Ci

p ou Cim :

1 chromosome sexuel Xm ou Xp

ovules

22 paires de chromosomes autosomaux homologues Ci

p ou Cim / Ci

p ou Cim

2 chromosomes sexuels Xm ou Yp/ Xm ou Xp

Enfant

246 = 1013 possibilités

Transmission des caractères parentaux chez l ’homme

61

Un gène

génotypephenotype

allèles

lignées puresA/A a/a F f

A/a A/a F F

A/A a/a A/a F f F0.25 0.25 0.5

hybride de 1ière génération

hybrides de 2de génération

Deux gènes

A/a B/b A/a B/b F G F G

hybride de 1ière génération

B/B B/b b/b

A/A FG FG Fg

A/a FG FG Fg

a/a fG fG fg

B/B B/b b/bA/A 1/16 1/8 1/16

A/a 1/8 1/4 1/8

a/a 1/16 1/8 1/16

indépendants

gènes portés par deux

chromosomes différents (ou éloignés cf

crossing-over)

B/B B/b b/bA/A 1/4 0 0

A/a 0 1/2 0

a/a 0 0 1/4

liésgènes portés par le même chromosome

AB/ab

B/B B/b b/bA/A 1/4-2e e e2

A/a e 1/2-2e2 e

a/a e2 e 1/4-2e

crossing-over

e : fréquence de crossing-over, dépend de la distance entre les gènes (cMg :: e = 0.01)

Génétique mathématique

62