Download DNA recombination

1 ADN  2 ADN
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
DNA replication is semi-conservative
M. Meselson & P Stahl Proc.
Nat. Ac. Sci. 1958
2
The cell cycle
G0
Mitosis
In the resting state
(G0), cells do not divide
Gap 2
Gap 1
DNA Synthesis
3
DNA synthesis is catalyzed by DNA-dependent DNA polymerases
DNA
polymerase
5’GGATC
dATP
dTTP
nucleotides
dCTP
dGTP
CCTAGGAATCTTGGAACCGGGCCC
primer
5’
PPi
PPi PPi
PPi
PPi PPi
GGATCCTTAGAACCTTGGCCCGGG
CCTAGGAATCTTGGAACCGGGCCC
template
 DNA polymerization takes place in the 5’ to 3’ direction
 DNA polymerase requires a template and a primer
dNTP
template strand
strand to be synthesized
Stryer et al. Biochemistry, Freeman Edt
4
DNA replication requires a primase to start
 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
dNTP
template strand
strand to be synthesized
5
Leading and lagging strands
RNA primase
Okazaki
fragments
Size of Okasaki fragments : eukaryotes 200 bp
Alberts et al. MBOC, Garland Edt
6
On the « leading strand », DNA is continuously synthesized
Replication
fork
primase
DNA helicase
NTP
3’
5’
RNA
primer
3’
DNAPol d
dNTP
DNA helicase
5’
7
On the « lagging strand », DNA is synthesized discontinuously
RNA
primer
NTP
DNAPol a
primase
5’
DNA helicase
3’
DNAPol e
dNTP
RNA
primer
5’
DNA helicase
3’
RNAse and
dNTP DNAPol e
RNA
primer
5’
DNA helicase
3’
ligase
5’
Replication
fork
RNA
primer
DNA helicase
3’
8
The core of the eukaryote replication complex
DNAPol d
DNAPol e
Linda B. Bloom, University of Florida
http://www.med.ufl.edu/IDP/BMB/bmbfacultypages/lindabloom.html
DNAPol a
primase
Movies 5.1 (Molecules and Complexes) and
5.4 (Cell functions) Mol. Biol. Cell
 Eukaryote cells possesses several DNA polymerases (> 15)
a
d
e
nucleus
nucleus
nucleus
250 kDa
170 kDa
260 kDa
DNA primase, lagging strand
leading strand
lagging strand, DNA repair
9
Main components of the DNA replication complex
The catalytic core
DNA polymerase a – primase
DNA polymerase d, e
Replication protein C*
Proliferating cell nuclear antigen (PCNA)
primer RNA synthesis
DNA synthesis, leading+lagging strands
load PCNA on DNA
sliding clamp ensuring processivity
Topoisomerase
Helicase*
Adjusts DNA supercoiling
Unwinds DNA into strands
Replication protein A
Flap endonuclease 1
Dna2
RNase H1
DNA ligase 1
single strand DNA binding protein
removes RNA 5’-flap
removes RNA
joins Okasaki fragments
The replisome
* uses ATP
Cyclin A, cyclin B1
Cyclin dependent kinase 1, 2 (CDK1, CDK2)
+ 11 other proteins…
Temporal regulation
Maga and Hübscher 1996 Biochemistry 35: 5764-5777
Waga and Stillman 1994 Nature 269: 207-212
Frouin et al. 2003 EMBO reports 4: 666-670
Hübscher and Yeon-Soo Seo 2001 Mol. Cells 12: 149-157
10
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 d
and e, Fen1/Dna2, Lig1 (+ 15 other proteins !)
At 3’ OH end : RFC displaces Pol-a and loads PCNA + Pold/e
At the flap structure :
RFA dissociates Pole from PCNA
PCNA recruits Fen1/Dna2 which cleaves the flap
structure
PCNA recruits Lig1 that joins the DNA fragments
 PCNA is also involved in DNA repair mechanisms
PDB 1AXC
Maga and Hübscher 2003
Journal of Cell Science 116: 3051-3060
11
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
GFP
PCNA
Essert et al. 2005 Mol. Cell Biol. 25 : 9350-59
12
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
13
Replication starts at replication origins
1. Activation
ORC : origin replication complex
MCM : minichromosome
maintenance complex
Replisome
2. Extension
3. Termination
 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)
14
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
15
Sources of DNA damage
Replication errors: DNA polymerase
frequency 1/107
Molecular damages to DNA:
Exogenous
Origin
DNA damage
sun (1h/day)
chemical
T-T dimers
adducts
(base modification)
single strand breaks
double strand breaks
6-8.104
102-105
Possible
repair
Y
N
2-4.104
?
Y
±
single strand breaks
adducts/breaks
adducts
genome integration
2-4.104
104
102
?
?
Y
Y
Y
N
?
radioactivity
(natural
background)
Endogenous
temperature
free radicals
metabolites
viruses
transposons
number/cell.day
16
DNA repair mechanisms
Damage type
Repair
Recognition
T-T dimers
Restriction
Adducts
Excision
Single strand breaks
Synthesis
Ligation
Double strand breaks
Excision
or direct ligation
Recombination
Ligation
17
The COMET assay to measure DNA damages
also called single cell gel
electrophoresis (SCGE)
18
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.
chemical to
be tested
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.
Inhibition
zone
Brian Krug: Ames Test: Chemicals to Cancer
growth ring
Strain # S. typhimurium Type of Mutation Detected
Strain Name
1
TA98
detect frame-shift mutations
2
TA100
detect base pair substitutions
3
TA102
detect excision repair
4
TA104
detect base-pair substitutions
5
TA1534
detect frame-shift mutation
6
TA1530
detect base pair substitutions
19
Exemple of repair : thymine dimers
(induced by UV light)
Tymine dimer repair
enzyme : specific DNA
endonuclease
20
Metabolism et carcinogenicity of Benzo[a]Pyrene
Benzo[a]pyrene is a product of incomplete
combustion at temperatures between 300
and 600 °C.
translocation to
the nucleus
aromatic
molecule
(L)
Aryl
hydrocarbon
Receptor
AhR
AhR-L
AhRE
AhR-L
AhRE
induction of specific
mRNA (AhRE)
benzo[a]pyrene (BP)
CYP1A1, CYP1A2
epoxide hydrolase
benzo[a]pyrene-7,8-dihydrodiol
-9,10-epoxide
P450 cytochromes (phase I) :
CYP1A1, CYP1A2, CYP1B1, CYP2S1
Phase II enzymes : GST, UGT
(detoxification mechanism)
the diol
epoxide
covalently
binds to
DNA
(adduct)
Growth
Differentiation
Metabolism
(toxicity)
Increased
DNA
mutations
& cancer
21
Shimizu et al. (2000) PNAS 97 : 779-782
Benzo[a]pyrene carcinogenicity is lost in mice
lacking the aryl hydrocarbon receptor
Individual susceptibility to xenobiotics.
Exemple of CYP genes
Dossier INSERM
Dioxines dans l’environnement. Quels risques
pour la santé ?
http://ist.inserm.fr/basisrapports/rapport.html
22
ADN1 + ADN2  ADN3 + ADN4
DNA recombination : programmed random
modifications of the genome
 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
23
1. Homologous recombination
Condition : presence of two homologous sequences in
adjacent chromosomes or DNA molecules
The mechanism of homologous recombination
homology
Holliday
junction
cleavage
1
exchange
cleavage
2
ligation
displacement
(branch
migration)
ligation
25
RecA proteins catalyze the exchange of DNA strands ...
Structure of a RecA
polymer
ATP hydrolysis
ATP binding site
26
… in the 5’ to 3’ direction
without RecA
with RecA
… dans un seul sens
Driving force : ATP hydrolysis
27
Recombination events in cells
Example
Cells
Effect
Effector proteins
Crossing-over
Meiotic cells
( germinal cells)
genome
RecA-D like
rearrangements proteins
Virus integration
Host cell genome
dormancy
lytic/lysogenic
phases
Integrase
Integration Host
Factor
Conjugation
Bacteria
gene exchange
Integrase
VDJ recombination
lymphocytes
antibody and
TCR diversity
Rag1-2
Transposons
all cells
genome
Transposases
rearrangements
28
Mitosis, meiosis and fecundation
example of a diploid organism with 2 pairs of homologous chromosomes
diploid
gametes
diploid
2 haploids
2 diploids
diploid
MITOSIS
4 haploids
FECUNDATION
MEIOSIS
29
Mitosis : 1 diploid -> 2 diploids
Mitotic
spindle
DNA
replication
separation of sister
chromatides
decondensation
of chromosomes
centromere
s
Chromosome
condensation
separation of
daughter cells
(cytokinesis)
Sister
chromatides
30
Meiosis : 1 diploid -> 4 haploids
synaptolemal
complex
DNA
replication
Pairing of
homologous
chromosomes
1st mitosis
separation of
homologous
chromosomes
centromere
Chromosome
condensation
gametes
Sister
chromatids
2nd mitosis
31
Recombination during meiosis
synaptolemal
complex
DNA
replication
Pairing of
homologous
chromatids and
crossing-over
1st mitosis
segregation of
homologous
chromosomes
centromer
Chromosome
condensation
gametes
sister
chromatids
2ndmitosis
32
Non-Mendelian transmission
« Crossing over »
simple
paternal chromosome
maternal chromosome
 homologous sequence
 frequency : 1/107 base pairs, at
least one per chromosome
double
Mitochondrial DNA transmission
 Exclusive transmission of mother mitochondria
Epigenetics
 Some genes are inactivated by methylation, the methylation state can be
transmitted to daughter cells.
 Example : inactivation of one chromosome X in women
33
Application of recombination : gene knock-out by insertion
blasticidineR
target gene
blasticidineR
Recombination
(double
crossing-over)
WT
ampicillineR
Dphg1a Dphg1b DDphg1a/b
PHG1A
Anti-PHG1A
PHG1B
Benzhegal et al. 2002
Anti-PHG1B
34
2. Site-specific recombination
Condition : presence of a specific sequence repeated twice
Mechanism : 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)
Example 1 : site-specific recombination of a virus
The two states of the bacteriophage l
Reversible recombination
DNA of the
bacterio
phage l
attP
DNA of E. coli
attB
Integrase
Integration
Host Factor
Excisionase
Integrase
Integration
Host Factor
Recombinant DNA
37
Integrase mechanism
phage l
DNA
attP
E. Coli DNA
attB
recombinant DNA
pairing,
double cleavage, double exchange,
ligation
38
phage DNA
attB
attP
bacterial
DNA
Conformation 1 : phage and bacterial DNA separated
Conformation 2 : phage and bacterial DNA fused
39
integration
Phage integration in bacterial genome
excision
Biswas et al. (2005) A structural basis for allosteric control of DNA recombination
by λ integrase Nature 435 : 1059-1066
40
Example 2. The F-factor allows gene exchange between bacteria
Conjugation
Reversible recombination
factor F
« female »
bacterial chromosome
integration
DNA
Hfr chromosome
excision
« male »
plasmide F ’
episome F
 F’ plasmids often carry virulence factors
41
Example 3 : genetic rearrangements in B lymphocytes
Example : light chain k of antibodies
recombination
splicing
RAG : recombination activating genes
RSS : recombination signal sequences
42
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 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 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 Vregion exon.
43
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 genome
Mechanism : specialized protein complex, no branch
migration, duplication of ends
Transposons are mobile DNA sequences in genomes
transposase
transcription
traduction
excision
insertion
example : Tn5 transposon and transposase
47
The presence of transposons allows gene duplication, inversion or
excision by homologous recombination
DELETION
INVERSION
DUPLICATION
48
Viruses and transposons
Transposons
Viruses
 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)
transcriptase
réverse
RNA viruses
cDNA
ARNm
reso lvase
transcrip tase
réverse
resolvase
type II transposons
acti vi té de
restriction
DNA viruses
ADN exci sé
acti vi té
d'i ntégration
transposase
ARNm
49
Fast and slow viruses
 Fast viruses
Productions de protéines et
Production of viral proteins
acides
nucléiques
and nucleic
acids,viraux
formation
par
la
cellule
et
enpaquetage
of new virus particle
de
nouveaux
Cell
death virus
Destruction de la cellule
entréeentry
du virus
Virus
by fusion of
the
virus
envelope
with
par fusion avec
the
plasma membrane
la membrane
plasmique
thanks
to
cell
receptors
gràce à des récepteurs
de la surface cellulaire
Pour
les viruses,
virus à ARN
For
RNA
a
reverse
transcriptase
copie en ADN par une
copy
their RNA réverse
into
transcriptase
DNA
virale
The
virus de
takes
control
Contrôle
la cellule
of the cell
 Slow viruses
Intégration dans le génome
Genome integration
Silence
expression
Silent
expression
Dormance
Dormancy
50
Transmission des caractères parentaux chez l ’homme
Père
Mère
 22 paires de chromosomes
autosomaux homologues Cip/Cim
2 chromosomes sexuels Xm/Yp
 22 paires de chromosomes
autosomaux homologues Cip/Cim
2 chromosomes sexuels Xm/Xp
spermatozoïdes
ovules
 22 chromosomes autosomaux
Cip ou Cim
1 chromosome sexuel Xm ou Yp
 22 chromosomes autosomaux
Cip ou Cim :
1 chromosome sexuel Xm ou Xp
Enfant
 22 paires de chromosomes autosomaux
homologues Cip ou Cim / Cip ou Cim
2 chromosomes sexuels Xm ou Yp/ Xm ou Xp
246 = 1013 possibilités
52
Génétique mathématique
Un gène
allèles
génotype
phenotype
Deux gènes
A/A
F
a/a
f
lignées pures
A/a
F
A/a
F
hybride de 1ière
génération
A/a B/b
F G
hybride de 1ière
génération
A/a B/b
F G
B/B
B/b
b/b
A/A
FG
FG
Fg
A/a
FG
FG
Fg
fG
fG
fg
A/A
F
a/a
f
A/a hybrides de
F
2de génération
a/a
0.25
0.25
0.5
indépendants
B/B
A/A
1/16
B/b
b/b
1/8
1/16
A/a
1/8
1/4
1/8
a/a
1/16
1/8
1/16
B/B
B/b
b/b
crossing-over
B/B
B/b
b/b
A/A
1/4-2e
e
e2
A/a
e
1/2-2e2
e
liés
a/a
e2
e
1/4-2e
A/A
1/4
0
0
A/a
0
1/2
0
a/a
0
0
1/4
e : fréquence de crossing-over, dépend de la
distance entre les gènes (cMg :: e = 0.01)
gènes portés
par deux
chromosomes
différents (ou
éloignés cf
crossing-over)
gènes portés
par le même
chromosome
AB/ab
53
L’ADN simple brin est généré par l ’action d ’une hélicase et d’une
endonuclease du complexe RecBCD
 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
base
Chez E. coli, la recombinaison est
catalysée par l ’action de quatres
protéines RecA, RecB, RecC et RecD
54