Recombination
Homolougous recombinationSite-specific recombination
Molecular mechanisms of gene transfer in bacteria and bacterial recombination
Construction of a recombinant DNA molecule
Cross-over duringmeiosis gives riseto recombinant chromosomes
Recombinação do material genéticoQualquer via pela qual dois elementos de ácido nucleico interactuam
tendo como resultado a alteração da sequência de, pelo menos, um deles
Recombinação homólogaImplica extensa homologia das moléculas que interactuam
Ex. Ocorre durante a meiose (reprodução sexuada)
Recombinação sítio-específicaEnvolve curtas sequências homólogas
Ex. Integração de fagos nos genomas bacterianos
“Transposição”Implica interacção das extremidades do elemento transponível com
sequências alvo na molécula hospedeiraEx. Integração de transposões
Mechanism of homologous recombination: the Holliday model of genetic recombination
Paired DNA molecules and nicks
Strand invasion
Ligation of nicks and branch migration
Branch migration increases sizes of heteroduplex regions
Resolution of Holliday intermediate
Several proteins, RuvA, RuvB and RuvC are responsible for establishing the crossand the cut to be made
Hollyday structure
Enzimologia da recombinação homóloga
• Várias enzimas envolvidas nas vias de recombinação homóloga.
• Diferentes vias (muitas vezes redundantes) que envolvem enzimascom actividade de nuclease, helicase.
• As vias, diferem em passos que precedem o emparelhamento e a troca de cadeias (ex. um corte em cadeia simples ou corte em cadeiadupla, uma só das moléculas de DNA ou em ambas) e nas diferentesenzimas envolvidas.
• Qualquer desta vias envolve emparelhamento e troca de cadeias de DNA, mediado por RecA (e auxiliado por SSB).
• As diferentes vias conduziram ao aparecimento de diferentes modelosque são variações ao modelo de Hollyday.
RecA protein causes single-stranded DNA to invade double-stranded DNA and to move along it until a
region of complementarity is found
3’-OH
Heteroduplex DNA polymeraseDNA ligase
The possible fates of transforming DNA
A donor wild-type allele A+ (cloned in a bacterial vector) transforms an A recipient by one of three different types of insertion
note: The recipient is generally of the same species as the donor DNA, either prokaryotic or eukaryotic
• Processo de grande importância biológica
Recombination between a linear moleculelinear molecule and a circular moleculecircular molecule.Recombination at two sites leads to replacement of a portion of the circular molecule
A linear molecule only integratesby a double-recombination event(at two sites)
Recombination between two circular moleculescircular molecules.Recombination at a single siteleads to integration of the plasmid or phage into the chromosome
Selection for single site (1 c.o) integration and
two site (2 c.o) integration
Media without X
Marker(ex. Antibiotic resistance gene)
Type ofSELEC
TION
Note: in order to increase de number of recombinants due to double crossover at 1 and 2, plasmid DNA should be linearized
kanR
Inactivated gene
T.P.CComo seleccionava este recombinante?
Two-step gene replacement
T.P.CQual a vantagem desta estratégia?
DNA transfer in bacteria
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* RecombinationSe após a transferencia não há recombinação perde-se a informaçãoGeralmete são processos não recíprocos
Transformation
● Competence B. subtilis 20%, several hours
Streptococcus 100%, few minutes
● Uptake DNA ex. Haemophilus (Gram -):- ds DNA entry- degradation to ss DNA in periplasmic space- ss integration
Bacillus and Streptococcus (Gram+):- ss DNA entry
Different DNA fragments sizes
Specific short DNA sequences for irreversible binding
A C
DB
DNA transfer by transformation
A detecção de recombinantes (organismos diferentes dos progenitores) faz-se através da utilização de mutantes, meios selectivos, marcadores de DNA
Isolation of different transformants depend on selection
MEDIA
B + C
A + C
A + B
C
Conjugation
Integration of plasmid F in the E. coli chromosome through homologous recombination at
IS (insertion sequences)
tra region
The genetic map of the F plasmid
oriT
IS3
Genetic Map of the Escherichia coli chromosome
Typical Insertion Sequence (IS)
Insertion sequences are simple transposable elements found in bacteria
Pierce
Stuctures of common insertion sequences
Pierce
Formation of an Hfr
oriTIS3
IS3
Homologous recombination (RecA dependent)
Integração tipo Campbell
Formation of different Hfr strains depends on the E. coli chromosome site of plasmid F integration
Breakage of an Hfr chromosome at the origin of transfer (oriT)
oriT
pro is the first gene to be transfered
Transfer of single-stranded fragment of donor chromosome, and recombination with recipient chromosome. note: double crossovers can occur in any location
Lytic and lysogenic cycle of bacteriophages
Generalized transduction
Transducing particle(host DNA within phage coat)
Lytic cycle
Selection of transductants and cotransductants
a and b are sufficiently closed to be cotransducted
Integration of the phage λ DNA into the E. colichromosome
Integration of the phage λ DNA into the E. colichromosome
Site-specific recombination:
- specific integration site (att)- depends on a specific integration enzyme (integrase coded by λ int gene)
(integrase)
Genetic map of phage λ
att siteint gene
xis gene
att sites present the phage DNA (att P) and bacteria chromosome (att B)
INTEGRATION
Prophage
attL attR
EXCISIONdepend on the λ gene products Xis (excisionase), Int and
IHF