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

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DNA replication is semi-conservative

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

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3

The cell cycle

Gap 1

DNA Synthesis

Mitosis

Gap 2

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

G0

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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 Edt4

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

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Okazaki fragments

RNA primase

Leading and lagging strands

Size of Okasaki fragments : eukaryotes 200 bp

Alberts et al. MBOC, Garland Edt6

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5’3’

dNTPRNA primer

5’3’

NTPprimase

Replication fork

DNAPol

DNA helicase

DNA helicase

On the « leading strand », DNA is continuously synthesized

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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

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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

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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

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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

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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

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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 : 9350-5913

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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

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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

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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 ? ? 16

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

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The COMET assay to measure DNA damages

also called single cell gel electrophoresis (SCGE)

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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

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Exemple of repair : thymine dimers

Tymine dimer repair enzyme : specific DNA endonuclease

(induced by UV light)

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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

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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

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DNA recombination : programmed random modifications of the genome

ADN1 + ADN2 ADN3 + ADN4

Molecular mechanisms of homologous recombination

Site specific recombination

Conjugation, mechanism of bacterial parasexuality

The VDJ recombination, one of the mechanisms that generate

antibody and TCR diversity

The crossing-over at meiosis increases genomic diversity in the

population

Transposons and viruses are mobile DNA/RNA sequences

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1. Homologous recombination

Condition : presence of two homologous sequences in adjacent chromosomes or DNA molecules

homology

cleavage 1

ligation

exchange

displacement(branch migration)

Holliday junction

cleavage 2

ligation

The mechanism of homologous recombination

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ATP binding site

ATP hydrolysis

RecA proteins catalyze the exchange of DNA strands ...

Structure of a RecA polymer

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… dans un seul sens

without RecA with RecA

Driving force : ATP hydrolysis

… in the 5’ to 3’ direction

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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

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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

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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

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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

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DNA replication

segregation of homologous

chromosomes

gametes

Chromosome condensationcentromer

sister chromatids

Pairing of homologous

chromatids and crossing-over

synaptolemal complex

1st mitosis

2ndmitosis

Recombination during meiosis

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« Crossing over »

Mitochondrial DNA transmission

Exclusive transmission of mother mitochondria

simple

double

homologous sequence frequency : 1/107 base pairs, at least one per chromosome

paternal chromosome

maternal chromosome

Epigenetics

Some genes are inactivated by methylation, the methylation state can be transmitted to daughter cells. Example : inactivation of one chromosome X in women

Non-Mendelian transmission

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ampicillineR

blasticidineR

target gene blasticidineR

Recombination (double

crossing-over)

WT

PHG1A

phg1a phg1bphg1a/b

PHG1B

Anti-PHG1B

Anti-PHG1A

Benzhegal et al. 2002

Application of recombination : gene knock-out by insertion

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2. Site-specific recombination

Condition : presence of a specific sequence repeated twiceMechanism : specialized protein complex, no branch migration

Specific case : recombination with a circular DNA molecule

• Simple recombination

• Double recombination

• Recombination with circular DNA : local double recombination (no branch migration)

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

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Integrase mechanism

phage DNA

E. Coli DNA

attP

attB

recombinant DNA

pairing, double cleavage, double exchange, ligation

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

attB attP

bacterial DNA

phage DNA

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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

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Conjugation Reversible recombination

« female »

« male »

DNA

episome F

factor F

bacterial chromosome

Hfr chromosome

plasmide F ’

integration

excision

F’ plasmids often carry virulence factors

Example 2. The F-factor allows gene exchange between bacteria

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Example : light chain of antibodies

Example 3 : genetic rearrangements in B lymphocytes

recombination

RAG : recombination activating genesRSS : recombination signal sequences

splicing

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In every V-region recombination event, the signals flanking the gene segments are brought together to allow recombination to take place. Immunobiology: The Immune System in Health and Disease. 5th Ed.Janeway CA et al. New York: Garland Science; 2001.

In some cases, as shown in the left panels, the V and J gene segments have the same transcriptional orientation. Juxtaposition of the recombination signal sequences results in the looping out of the intervening DNA. Heptamers are shown in orange, nonamers in purple, and the arrows represent the directions of the heptamer and nonamer recombination signals. Recombination occurs at the ends of the heptamer sequences, creating a signal joint and releasing the intervening DNA in the form of a closed circle. Subsequently, the joining of the V and J gene segments creates the coding joint.

In other cases, illustrated in the right panels, the V and J gene segments are initially oriented in opposite transcriptional directions. Bringing together the signal sequences in this case requires a more complex looping of the DNA. Joining the ends of the two heptamer sequences now results in the inversion and integration of the intervening DNA. Again, the joining of the V and J segments creates a functional V-region exon.

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 44

Inducible tissue specific promoter

Mating

3. Transposon and viruse integration in the genome

Condition : random (?) integration in the genomeMechanism : specialized protein complex, no branch migration, duplication of ends

Transposons are mobile DNA sequences in genomes

excision insertiontranscriptiontraduction

transposase

example : Tn5 transposon and transposase 47

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

DELETION INVERSION

DUPLICATION

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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 de restriction activité

d'intégration

ADN excisé

DNA viruses

RNA viruses

Viruses and transposonsTransposons Viruses

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entrée du virus par fusion avecla membrane plasmique gràce à des récepteurs de la surface cellulaire

Pour les virus à ARN copie en ADN par une transcriptase réverse virale Contrôle de la cellule Intégration dans le génome

Silence expression Dormance

Productions de protéines etacides nucléiques virauxpar la cellule et enpaquetage de nouveaux virus Destruction de la cellule

Fast viruses

Slow viruses

Fast and slow viruses

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Virus entry by fusion of the virus envelope with the plasma membrane thanks to cell receptors

For RNA viruses, a reverse transcriptase copy their RNA into DNAThe virus takes control of the cell

Production of viral proteins and nucleic acids, formation of new virus particleCell death

Genome integrationSilent expressionDormancy

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

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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

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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

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