The second sectionThe second section of of recombination systemsrecombination systems

SS ite-Specific Recombination ite-Specific Recombination & &

Transposition Transposition

By: Hatice Nur HalipçiBy: Hatice Nur Halipçi

OUTLINEOUTLINE

Site-Specific RecombinationSite-Specific Recombination Biological Roles of Site-Specific Biological Roles of Site-Specific

RecombinationRecombination TranspositionTransposition Phage Mu- Phage Mu- a model transposable a model transposable

elementelement

Two classes of genetic Two classes of genetic recombinationrecombination

SSite-specific recombination (SSR)ite-specific recombination (SSR) Transpositional recombinationTranspositional recombination

Site-Specific RecombinationSite-Specific Recombination (SSR)(SSR)

SSRSSR is is recombination between two recombination between two defined sequence elementsdefined sequence elements

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

SSR can generate three different types of SSR can generate three different types of DNA rearrangements:DNA rearrangements:

1.Insertion1.Insertion

2.Deletion2.Deletion

3.Inversion3.Inversion

Insertion is the addition of one or more nucleotide base pairs into a DNA sequence

Deletion (also called deficiency) is a mutation in which a part of a chromosome or a sequence of DNA is missing. Inversion is a chromosome rearrangement in which a segment of a chromosome is reversed end to end, shortly reversing the orientation of a chromosomal segment.

Structures involved in SSRStructures involved in SSR

1-2 Site-specific recombinases cleave and rejoin DNA

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

1. 1. SerineSerine Recombinases Recombinases

2. 2. TyrosineTyrosine Recombinases 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 fFirst , the serine recombinases cleave all four strandsour strands

Second, DNA swap occursSecond, DNA swap occurs Finally, the serine recombinases are liberaFinally, the serine recombinases are libera

ted and they seal the DNA strandsted 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 time

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

Recombination by a tyrosine reco

mbinase

Biological roles of site-specific Biological roles of site-specific recombinationrecombination

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

Bacteriophage Bacteriophage infects a host bacterium and infects a host bacterium and would establish a would establish a lysogenlysogen ( (staying in the host staying in the host

chromosome presently without harmingchromosome presently without harming)) ,which req ,which requires the integration of phage DNA into host chuires the integration of phage DNA into host chromosomeromosome

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

attPattP site is on the phage DNA and site is on the phage DNA and attBattB site is site is on the bacterial genomeon the bacterial genome

IntInt is a is a type of type of tyrosine recombinasetyrosine recombinase

The highly asymmetric organizatiThe highly asymmetric organization of the on of the attPattP and attB sites is impand attB sites is important to the regulation of ortant to the regulation of integra integra

tiontion

The figure showing:The figure showing:recombination sites involverecombination sites involved in integration and excisiod in integration and excision showing the important sen showing the important sequence elementquence element

2-2 2-2 Phage Phage excision requires a excision requires a new DNA-binding proteinnew DNA-binding protein

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

Xis binds to the integrated Xis binds to the integrated attRattR sites to sti sites to stimulate excision and to inhibit integrationmulate 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 The Salmonella Hin recombinaseSalmonella Hin recombinase inverts inverts

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

Hin recombinase is an example of prograHin recombinase is an example of programmed rearrangements in bacteriammed rearrangements in bacteria

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

Hin is a Hin is a serineserine recombinase which promot recombinase which promotes inversiones inversion

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

Hin recombination requires a DNA enhaHin recombination requires a DNA enhancerncer in addition to the hix sites in addition to the hix sites

Enhancer function Enhancer function requires the bacterial requires the bacterial Fis protein Fis protein

the enhancer-Fis complex activates the cthe enhancer-Fis complex activates the catalytic steps of recombination atalytic steps of recombination

Hin-catalyzed inversion is not highly regulHin-catalyzed inversion is not highly regulated, rather, inversion occurs stochasticallated, rather, inversion occurs stochasticallyy

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

CCircular DNA molecules sometimes form ircular DNA molecules sometimes form dimers and even higher multimeric forms ddimers and even higher multimeric forms during the process of homologous recombinuring the process of homologous recombination ation

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

Circular DNA molecules can form multimers

Xer recombinase is a tyrosineXer recombinase is a tyrosine (site-spesific (site-spesific recombinase)recombinase)

Xer catalyzes the monomerization of bacterial cXer catalyzes the monomerization of bacterial chromosomes and of many bacterial plasmidshromosomes and of many bacterial plasmids

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

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

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

Pathways for Xer-mediated recombination

TranspositionTransposition

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

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

These mobile genetic elements are called These mobile genetic elements are called transposable elements or transposonstransposable elements or transposons

Transposition of a mobile genetic Transposition of a mobile genetic element to a new site in DNAelement to a new site in DNA

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

They can also insert within the regulatory sequeThey can also insert within the regulatory sequences of a gene where their presence may lead to nces of a gene where their presence may lead to cchanges in how that gene is expressedhanges in how that gene is expressed

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

The recombinase responsible for transpositiThe recombinase responsible for transposition are usually calledon are usually called transposasestransposases or ,sometior ,sometimes,mes,integrasesintegrases

Transposons exist as both autonomous and Transposons exist as both autonomous and nonautonomousnonautonomous elementselements

For example: For example: IS10R is an IS10R is an autonomousautonomous elementelement while IS10L is while IS10L is non-autonomousnon-autonomous

NNonautonomousonautonomous transposons need a helper transposons need a helper transposon to mobilizetransposon to mobilize

Types of TranspositionTypes of Transposition

Cut and pasteCut and pasteTranspositionTransposition((ConservativeConservative))

Replicative transpositionReplicative transposition

RetrotranspositionRetrotransposition

CCut-and-ut-and-PPasteaste transposition transposition (conservative) (conservative)

In In cut-and-pastecut-and-paste transposition transposition, an element is cut out of one, an element is cut out of one site in a chromosome and pasted into a new site.site in a chromosome and pasted into a new site.

Replicative transpositionReplicative transposition

In In replicative transpositionreplicative transposition, an element is replicated, and one, an element is replicated, and one

copy is inserted at a new site; one copy also remains at the originalcopy is inserted at a new site; one copy also remains at the original

site.site.

RetrotranspositionRetrotransposition

In In retrotranspositionretrotransposition, , tthe element makes an RNA copy of itselfhe element makes an RNA copy of itself which is which is reversed-transcribedreversed-transcribed into a DNA copy which is then into a DNA copy which is then inserted (cDNAinserted (cDNA))

3-3-22 There are three principle classes There are three principle classes of transposable elementsof transposable elements

·· DNA transposonsDNA transposons carry carry terminal inverted terminal inverted repeats at both sites (repeats at both sites (recognize recombinase)recognize recombinase)

·· These inverted repeats act as the substrates These inverted repeats act as the substrates fofor r recombination reactions mediated by the recombination reactions mediated by the transposasetransposase

·· TheyThey carry a gene encoding their own transpos carry a gene encoding their own transposase, sometimes they may carry a few additionase, sometimes they may carry a few additional genesal genes which useful for the host (resistance which useful for the host (resistance etc.)etc.)

Viral-like retrotransposonsViral-like retrotransposons and retroviruses car and retroviruses carry ry terminal repeat sequencesterminal repeat sequences and two genes im and two genes important for recombinationportant for recombination

Viral-like retrotransposons Viral-like retrotransposons and retroviruses carand retroviruses carry ry LTRsLTRs (long terminal repeats)(long terminal repeats)

,,

Viral-like retrotransposonsViral-like retrotransposons encode two proteins encode two proteins needed for their mobility: needed for their mobility: integraseintegrase andand reverse reverse transcriptase (RTtranscriptase (RT))

Poly-A retrotransposonsPoly-A retrotransposons look like genes look like genes

They carry They carry 5’ UTR ve 3’ UTR (no translation here)

And also carry an A-T sequence ORF1 encodes the protein which binds

RNA ORF2 encodes the protein which activates

reverse transcriptase and endonuclease

BBacteriophageacteriophage--MuMu

a model transposable a model transposable elementelement

Temperate bacteriophage Mu (MuTemperate bacteriophage Mu (Mu--mutator)mutator) Upon infection of its E.coli host, phage Mu integrates by Upon infection of its E.coli host, phage Mu integrates by

conservative transpositionsconservative transpositions Mu genome is ~ Mu genome is ~ 37 kb 37 kb In the Mu system, transposition is obligatory for replication In the Mu system, transposition is obligatory for replication

an thus the phage genome is replicated only as a an thus the phage genome is replicated only as a cointegrate structurecointegrate structure

The biochemistry of Mu transpositionThe biochemistry of Mu transposition

The transposase is a protein called The transposase is a protein called MuA.MuA. It It has both Dna binding and endonuclease has both Dna binding and endonuclease activitiesactivities

MuA normally acts in a multiprotein complex MuA normally acts in a multiprotein complex with several accessory proteinswith several accessory proteins

MuBMuB is an activator of MuA and provides is an activator of MuA and provides some degree of target-site selectivity some degree of target-site selectivity

HUHU is an E. Coli non-sequence –specific is an E. Coli non-sequence –specific DNA binding proteinDNA binding protein