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. - Durante a meiose (reprodução sexuada)- Mecanismos de reparação do DNA

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 enzimas com actividade de nuclease, helicase.

• Qualquer desta vias envolve emparelhamento e troca de cadeias de DNA, mediado por RecA (e auxiliado por SSB).

• As vias, diferem em passos que precedem o emparelhamento e a troca de cadeias (ex. um corte em cadeia simples ou corte em cadeia dupla, uma só das moléculas de DNA ou em ambas) e nas diferentes enzimas envolvidas.

• As diferentes vias conduziram ao aparecimento de diferentes modelos que sãovariações ao modelo de Hollyday.

• As duas regiões de DNA que recombinam têm de ser muito semelhantes masnão necessariamente iguais.

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

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

Recombination at two sitesleads to to replacement of aportion of the circular molecule

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?

Mechanisms of gene transfer

in bacteria and viruses useful in recombination studies

DNA transfer in bacteria

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* RecombinationSe após a transferencia não há recombinação perde-se a informaçãoGeralmente são processos não recíprocos

Conjugationex. F plasmid in E. coli

Formation of a mating pair

Integration of plasmid F in the E. coli chromosome through homologous recombination at

IS (insertion sequences)

tra

regi

on

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

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 Transfer from a Hfr strain starts from adefined point and proceeds in a specific direction

Chromosomal transferred genes depend on the site of insertion of F plasmid:

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

Mapa genético do cromossoma de E. coli: - as unidades de distância são definidas como minutos de transferência do cromossoma(cada minuto corresponde a aprox. 47 000 bases transferidas)

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

Transduction

Lytic and lysogenic cycle of bacteriophages

Lysogenic bacteriophage(temperate phage)

integration of the phage λDNA into the E. coli chromosome

occurs via a site-specific recombination event

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)

cos cos

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

Aberrant excision

The progeny phage producedwill all contain this bacterial gene

This gene will be transduced at a veryhigh transduced frequency but limitedto a small region of the chromosome

Lytic phages

Generalized transduction

Transducing particle(host DNA within phage coat)

Lytic cycle

Homologous recombination

Selection of transductants and cotransductants

a and b are sufficiently closed to be cotransducted