BIO353 – lecture 7: Conservative site-specific recombination

BIO353.01 – Molecular Genetics shf – 2022

 Recombination: intra- vs intermolecular

intermolecular
exchange of
single crossover chromosome arms

similar sequence

exchange of
DNA fragment
double crossover

intramolecular
similar sequence excision of
DNA fragment
direct repeats

similar sequence

inverted repeats
inversion of
DNA fragment

BIO353.01 – Molecular Genetics shf – 2022

 Recombination: integration of viral DNA into the host genome

some viruses use a transposition-like recombination mechanism to integrate into host genomes

BIO353.01 – Molecular Genetics shf – 2022

 Recombination: site-specific vs transposition

site-specific
recombination

transposition

target DNA

BIO353.01 – Molecular Genetics shf – 2022

 Recombination: modes of recombination - outcomes

insertion excision inversion

recombination
recombination
deletion

recombination
inversion

insertion

BIO353.01 – Molecular Genetics shf – 2022

 Recombination: general mechanism

loxP (locus of crossing over in P1) sequence

non-palindromic
crossover region
cleavage site

5’-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3’
3’-TATTGAAGCATATCATATGTAATATGCTTCAATA-5’
cleavage site
recombinase binding site recombinase binding site

palindromic
repeat

BIO353.01 – Molecular Genetics shf – 2022

 Recombination: general mechanism

recombinase binding sites

T AGC
ATC G
recombnination
partners T AGC
ATC G
crossover
region
recombinase binds

recombinase

T AGC
ATC G

T AGC
ATC G

strand exchange

T AGC
ATC G
recombnination
product T AGC
ATC G

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: form covalent intermediates with DNA

the reaction is reversible without expenditure of energy

à the bond energy is conserved

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: two types – two mechnisms

1 step reaction
double strand exchange
rotation of DNA fragments

2 step reaction
single strand exchange
Holliday junction

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Serine recombinases

crossover region

recombinase recognition sites

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Serine recombinases

binding of serine recombinase and cleavage of substrate

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Serine recombinases

swapping of DNA strands and rejoining

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Serine recombinases

joining of exchanged strands

crossover region

recombinase recognition sites

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Serine recombinases

DNA

DNA

recombinase dimer

rotation plane

recombinase dimer

DNA DNA

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Serine recombinases

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Tyrosine recombinases

binding of tyrosine recombinase subunits and single strand cleavage

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Tyrosine recombinases

exchange of top strand and formation of a Holliday junction

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Tyrosine recombinases

cleavage of bottom strand

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Tyrosine recombinases

cleavage of bottom strand

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Tyrosine recombinases

bottom strand exchange and resolution of Holliday junction

crossover region

recombinase recognition sites

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: two types - comparison

serine recombinases tyrosine recombinases

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Cre recombinase - a tyrosine recombinase

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: Cre recombinase - a tyrosine recombinase

BIO353.01 – Molecular Genetics shf – 2022

 Cre recombinase: and conditional knock-out animals

happy mouse lethal mutation

E1 E2 E3 E1 E3
promoter gene of interest promoter knocked-out gene
(expressed in all tissues) (exon 2 removed)

BIO353.01 – Molecular Genetics shf – 2022

 Cre recombinase: and conditional knock-out animals - flox system

happy mouse

E1 E2 E3
heart-specific
promoter gene of interest Cre recombinase
(expressed in all tissues) promoter (expressed only in heart)

loxP sites

BIO353.01 – Molecular Genetics shf – 2022

 Cre recombinase: and conditional knock-out animals - flox system

mating
happy mouse

E1 E2 E3
heart-specific
promoter gene of interest Cre recombinase
(expressed in all tissues) promoter (expressed only in heart)

loxP sites

all tissues except heart in heart tissue

E1 E2 E3 E1 E3
promoter intact gene promoter knocked-out gene
(exon 2 removed)

loxP site

conditional (heart-specific) knock-out

BIO353.01 – Molecular Genetics shf – 2022
 Cre recombinase: and conditional knock-out animals

mating
happy mouse
estrogen receptor sequence

E1 E2 E3
heart-specific
promoter gene of interest CreERT
(expressed in all tissues) promoter (expressed only in heart)

loxP sites

all tissues except heart in heart tissue

E1 E2 E3 E1 E3
promoter intact gene promoter knocked-out gene
(exon 2 removed)

estrogen (or tamoxifen)

conditional (heart-specific) knock-out

BIO353.01 – Molecular Genetics shf – 2022
 Cre recombinase: and conditional knock-out animals - flex system

gene / promoter

BIO353.01 – Molecular Genetics shf – 2022

 Cre recombinase: and conditional knock-out animals - flex system

cleavage site

5’-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3’
loxP site
3’-TATTGAAGCATATCATATGTAATATGCTTCAATA-5’
cleavage site
recombinase binding site recombinase binding site

5’-ATAACTTCGTATAAAGTATCCTATACGAAGTTAT-3’
lox2272 site
3’-TATTGAAGCATATTTCATAGGATATGCTTCAATA-5’

recombinase binding site recombinase binding site

BIO353.01 – Molecular Genetics shf – 2022

 Cre recombinase: and conditional knock-out animals - flex system

lox2272 loxP lox2272 loxP

gene / promoter

1st recombination
(inversion)

gene / promoter

BIO353.01 – Molecular Genetics shf – 2022

 Cre recombinase: and conditional knock-out animals - flex system

lox2272 loxP lox2272 loxP

gene / promoter

1st recombination
(inversion)

gene / promoter

2nd recombination
(excision)

gene / promoter

BIO353.01 – Molecular Genetics shf – 2022

 Cre recombinase: and conditional knock-out animals - flex system

lox2272 loxP lox2272 loxP

gene / promoter

1st recombination
1st recombination (inversion)
(inversion)

gene / promoter gene / promoter

2nd recombination
2nd recombination
(excision)
(excision)

gene / promoter gene / promoter

BIO353.01 – Molecular Genetics shf – 2022

 l phage: lysogenic cycle

lytic growth lysogenic state

site-specific
recombination

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: l integrase

l phage integration is mediated by recombination

l integrase
binding sites

attP
phage DNA

X crossover region

bacterial DNA
attB
l integrase
binding sites

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: l integrase

attP contains additional l binding sites

right arm attP left arm

phage DNA

X crossover region

bacterial DNA
attB
l integrase
binding sites

BIO353.01 – Molecular Genetics shf – 2022

 Recombinases: l integrase and architectural proteins

IHF (integration host factor)

weak binding

strong binding
BIO353.01 – Molecular Genetics shf – 2022
 Recombinases: l integrase - mechanism of integration

IHF (integration host factor)

integration excision

Xis recruits l binding to P2

BIO353.01 – Molecular Genetics shf – 2022
 Salmonella: immune system evasion

BIO353.01 – Molecular Genetics shf – 2022

 Salmonella: immune system evasion

H2 flagellin repressor
of H1 flagellin
expression

BIO353.01 – Molecular Genetics shf – 2022

 Salmonella: Hin recombination

Fis: factor of inversion stimulation

BIO353.01 – Molecular Genetics shf – 2022

 Salmonella: other uses of recombination

resolvase

BIO353.01 – Molecular Genetics shf – 2022