Chapter 11Chapter 11 Site-Specific Recombination & Site-Specific Recombination &

Transposition of DNATransposition of DNA

Two classes of genetic recTwo classes of genetic recombinationombination

• Conservative site-specific recombination (CSSR)

• Transpositional recombination

OUTLINEOUTLINE1. Conservative Site-Specific Reco

mbination2. Biological Roles of Site-Specific

Recombination3. Transposition4. Examples of Transposable Ele

ments and Their Regulation5. V(D)J Recombination

Conservative Site-Specific ReConservative Site-Specific Recombination(CSSR)combination(CSSR)

CSSR is recombination between two defined sequence elements

1-1 CSSR occurs at specific DNA sequences in the target DNA

CSSR can generate three different types of DNA rearrangements:

1.Insertion

2.Deletion

3.Inversion

Structures involved in CSSRStructures involved in CSSR

1-2 Site-specific recombinases cleave and rejoin DNA using a covale

nt protein-DNA intermediate

There’re two families of conservative site-specific recombinases:

1. Serine Recombinases

2. Tyrosine Recombinases

1-3 Serine recombinases introduce double-stranded breaks in DNA and then swap strands to promote recombi

nation

• First , the serine recombinases cleave all four strands

• Second, DNA swap occurs

• Finally, the serine recombinases are liberated and they seal the DNA strands

Recombination by a serine rec

ombinase

1-4 Tyrosine recombinases break and rejoin one pair of DNA strands at a ti

me

• In contrast to the serine recombinases ,the tyrosine recombinases cleave and rejoin two DNA strands first, and only then cleave and rejoin the other two strands.

Recombination by a tyrosine reco

mbinase

1-5 Structure of tyrosine recombinases bound to DNA reveal the mechanis

m of DNA exchange

• Cre is an enzyme encoded by phage P1 , which functions to circularize the linear phage genome during infection

• The recombination sites of Cre on the DNA are called lox sites.

• Only Cre protein and the lox sites are needed for complete recombination

Biological roles of site-specific rBiological roles of site-specific recombinationecombination

2-1 integrase promotes the integration and Excision of a Viral Genome into the Host Cell Chromosome

• Bacteriophage infects a host bacterium and would establish a lysogen ,which requires the integration of phage DNA into host chromosome

• To integrate, Int catalyzes recombination between two specific sites—attachment (att) sites

• attP site is on the phage DNA and attB site is on the bacterial genome

• Int is a tyrosine recombinase, and the mechanism of strand exchange follows the pathway described above for the Cre protein

The highly asymmetric organizatiThe highly asymmetric organizationon of the of the attPand attB sites is impoattPand attB sites is important to the regulation of rtant to the regulation of integrati integrati

onon

The following figure showing:

recombination sites involved in integration and excision showing the important sequence element

2-2 2-2 Phage Phage excision requires a neexcision requires a new DNA-binding proteinw DNA-binding protein

• Phage excision requires an architectural protein called Xis, which is phage-encoded

• Xis binds to the integrated attR sites to stimulate excision and to inhibit integration

2-32-3 The Hin recombinaseThe Hin recombinase inverts a seginverts a segment of DNA allowing expression of alment of DNA allowing expression of al

ternative genesternative genes• The Salmonella Hin recombinase inverts

a segment of the bacterial chromosome to allow expression of two alternative sets of genes

• Hin recombinase is an example of programmed rearrangements in bacteria

• In the case of Hin inversion,recombination is used to help the bacteria evade the host immune system

• Hin is a serine recombinase which promotes inversion

2-4 Hin recombination requires a D2-4 Hin recombination requires a DNA enhancerNA enhancer

• Hin recombination requires a DNA enhancer in addition to the hix sites

• Enhancer function requires the bacterial Fis protein

• the enhancer-Fis complex activates the catalytic steps of recombination

• Hin-catalyzed inversion is not highly regulated, rather, inversion occurs stochastically

2-5 2-5 RecombinaseRecombinase converts multimeric converts multimeric circular DNA molecules into monomerscircular DNA molecules into monomers

• circular DNA molecules sometimes form dimers and even higher multimeric forms during the process of homologous recombination

• Site-specific recombinases (sometimes called resolvases) can resolve dimers and larger multimers into monomers

Circular DNA molecules can form multimers

• Xer recombinase is a tyrosine

• Xer catalyzes the monomerization of bacterial chromosomes and of many bacterial plasmids

• Xer is a heterotetramer, containing two subunits of XerC and two subunits of XerD

• XerC and XerD recognize different sequence• The directional regulation of Xer-mediated reco

mbination is achieved through the interaction between the Xer recombinase and a cell diversion protein called FtsK

Pathways for Xer-mediated recombination at dif

2-6 There are other mechanisms to d2-6 There are other mechanisms to direct recombination to specific segairect recombination to specific sega

ments of DNAments of DNA

• The gene rearrangements responsible for assembly of gene segments encoding critical proteins for the vertebrate immune system—known as V(D)J recombination—also occurs at specific sites

TranspositionTransposition

3-1 Some genetic elements move to new chromosomal locations by transposition

• Transposition is a specific form of genetic recombination that moves certain genetic elements from one DNA site to another

• These mobile genetic elements are called transposable elements or transposons

Transposition of a mobile genetic eleTransposition of a mobile genetic element to a new site in host DNAment to a new site in host DNA

• The transposons can insert within genes or regulatory sequence of a gene, which results in the completely disruption of gene function

• They can also insert within the regulatory sequences of a gene where their presence may lead to shanges in how that gene is expressed

• Transposable elements are present in the genomes of all life-forms. (1) transposon-related sequences can make up huge fractions of the genome of an organism. (2) the transposon content in different genomes is highly variable

3-2 3-2 There are three principle classes There are three principle classes of transposable elementsof transposable elements

•

3-3 · The recombinase responsible for transposition are usually called transposases or ,sometimes,integrases

• DNA transposons carry a transposase gene, flanked by recombination sites

• DNA transposons carry a gene encoding their own transposase, sometimes they may carry a few additional genes

3-4 · Transposons exist as both autonomous and nonautonomous elements(Autonomous transposons and Nonautonomous transposons)

3-5 · Viral-like retrotransposons and retroviruses carry terminal repeat sequences and two genes important for recombination

• Viral-like retrotransposons and retroviruses carry LTRs

• Viral-like retrotransposons encode two proteins needed for their mobility: integrase and reverse transcriptase (RT)

3-6 · Poly-A retrotransposons look like genes

3-7 DNA transposition by a cut-a3-7 DNA transposition by a cut-and-paste mechanism nd-paste mechanism

• The movement of a DNA transposon by a non-replicative mechanism called cut-and-paste transposition

1.First , transposase binds to the terminal inverted repeats at the end of the transopon and brings the two ends of the transopon DNA together to generate a stable protein-DNA complex called the synaptic complex or transpososome

2.Next, the transopon DNA is excised from its original location in the genome

3.Then, the 3’-OH ends of the transopon DNA attack the DNA phosphodiester bonds at the site of the new insertion, this DNA segment is called the target DNA

4.At last, the transopon DNA is covalently joined to the DNA at the target site by DNA strand tranfer. This reaction introduced a nick into the target DNA

The cut-and-paste mechanism of trans

position

• The intermediate in cut-and-paste transposition is finished by gap repair

1.Two introduced nick are filled by a DNA repair polymerase(encoded by the host cell) and a DNA ligase

2.Filling in the gap gives rise to the target site duplications that flank transposons

• There are multiple mechanisms for cleaving the nontransferred strand during DNA transposition

1.An enzyme other than tranposase can be used to cleave the nontransfered strand

2.The ranposase itself cleave the nontransfered strand by using an unusual DNA transesterification mechanism

3.DNA cleavage via a transesterification reaction can also occur between two ends of the transposon

3-8 DNA transposition by a replicati3-8 DNA transposition by a replicative mechanismve mechanism

• First, the transposase protein is assembled on the two ends of the transposon DNA to generate a transpososome

• Then, DNA is cleaved at the ends of the transposon DNA

• Then, the 3’OH ends of the trsnsposon DNA are joined to the target sites by the DNA strand transfer reaction, which generate a doubly branched DNA molecule

• At last, The two DNA branches within this intermediate have the structure of a replication fork, and the DNA synthesis is proceeded

• This replication reaction generates two copies of the transposon DNA

3-9 3-9 Viral-like Viral-like rretrotransposons etrotransposons and and rretroviruses etroviruses move using an move using an RNA intermediateRNA intermediate

• Recombination for retroelements involves an RNA intermediate

1. A cycle of transposition starts with transcription of the retrotransposon (or retroviral) DNA sequence into RNA by cellular RNA polymerase. Transcription initiates at a promoter sequence within one of the LTRs.

2. The RNA is then reverse transcribed to generate the cDNA

3. The cDNA is recognized by Integrase and recombinate with a new target DNA site

4. Integrase assembles on the ends of this cDNA and cleaves a few nucleotides off the 3’ ends of each strand

5. Integrase catalyzes the insertion of cleaved 3’ ends into a DNA target site in the host cell genome using the DNA strand transfer reaction.

6. Gap repair reaction generates target-site duplications.

Mechanism of retroviral integration and transposition of viral-like retrotranspos

ons

3-10 DNA transposases and retroviral 3-10 DNA transposases and retroviral integrases are members of a protein integrases are members of a protein

superfamilysuperfamily

• Many different tranposases and integrases carry a catalytic domain that has a common three-dimensional shape

• This domain contains two D and a E • The tranposase/integrase proteins use this same

site to catalyze both the DNA cleavage and the DNA strand transfer

• Tranposases and integrases are only active when assembled into a synaptic complex, also called a transpososome, on DNA

3-10 3-10 Poly-A RetrotranspositionPoly-A Retrotransposition move move by a “by a “reverse splicingreverse splicing” mechanism” mechanism

• The Poly-A Retrotransposons use an RNA intermediate but use a mechanism different than that used by the viral-like elements. This mechanism is called target site primed reverse transcription

1. First, the DNA of an integrated element is transcripted by a cellular RNA polymerase

2. Then, newly synthesized RNA is exported to cytoplasm to produce ORF1 and ORF2 proteins

3. The protein-RNA complex then reenters the nuclease and associates with the cellular DNA

4.The endonuclease initiates the intergration reaction by introducing a nick in the chromosomal DNA

5. The 3’OH DNA end generated by the nicking action then serves as the primer for reverse transcription of the element RNA

Transposition of Transposition of a poly-A retrotraa poly-A retrotransposon by targnsposon by target site-primed reet site-primed reverse transcriptiverse transcripti

onon

Examples of transposable enements Examples of transposable enements and their regulationand their regulation

• Two types of regulation appear as recurring themes:

1. Trnasposons control the number of their copies present in a given cell

2. Trnasposons control target site choice

4-1 IS4-family transposons are compact el4-1 IS4-family transposons are compact elements with multiple mechanisms for copy ements with multiple mechanisms for copy

number controlnumber control

• Tn10 transposes via the cut-and paste mechanism, using the DNA hairpin strategy to cleave the nontransfered strands

• Tn10 limits its copy number in any given cell by strategies that restrict its transposition frequency. One mechanism is the use of an antisense RNA to control the expression of the transposase gene

• By this mechanism, cells that carry more copes of Tn10 will transcribe more of the antisense RNA, which in turn will limit expression of the transposase gene. The transposition frequencywill,therefore, be very low in such a strain

Antisense rAntisense regulation oegulation of Tn10 exprf Tn10 expr

essionession

4-2 Transposition is coupled to cellul4-2 Transposition is coupled to cellular DNA replicationar DNA replication

• Bacteria methylate their DNA at GATC sites and GATC sites are hemimethylated for a few minutes

• It is during the brief period—when the Tn10 DNA is hemimethylated—that transposition is more likely to occur

• Both RNA polymerase and transposase bind more tightly to the hemimethylated sequences than to their fully methylated versions. As a result, when the DNA is hemimethylated, the transposase gene is most efficiently expressed, and the transposaseprotein binds most efficiently to the DNA

Transposition Transposition of Tn10 after of Tn10 after

passage of a rpassage of a replication foreplication for

kk

V(D)J recombinationV(D)J recombination

• The principal mechanism cells use to generate antibodies and T cell receptors with such diversity relies on a specialized set of DNA rearrangement reactions known as V(D)J recombination

Overview of tOverview of the process of he process of V(D)J recombV(D)J recomb

inationination

The early events in V(D)J recombinatiThe early events in V(D)J recombination occur by a mechanism similar to tron occur by a mechanism similar to tr

ansposon excisionansposon excision

• Revombination sequences, called recombination signal sequences, flank the gene segments that are assembled by V(D)J recombination

• Recombination always occurs between a pair of recombination signal sequences which are organized as inverted repeats flanking the DNA segments that are destined to be joined

• First, reconbinase recognizes the recombination signal sequences and pairs the two sites to form a protein-DNA synatic complex

• Then, the RAG1 proteins within this complex introduce single-stranded breaks in the DNA at each of the junctions between the recombination signal sequence and the gene segment that will be rearranged

• Then, this 3’OH DNA end attacks the opposite strand of the DNA double helix,which results in the generation of a hairpin DNA end

The The V(D)J recoV(D)J recombination pathmbination path

wayway