308 Lecture 15

308 Lecture 15 - Genetic recombination 1. General or...

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Genetic recombination 1. General or homologous recombination Genetic exchange between a pair of homologous DNA sequences 2. Site-specific recombination : occurs between sequences with a limited stretch of similarity; involves specific sites (recombination sites) 3. Transposition : mobile DNA element moves from one site to another (donor and target site), usually little sequence similarity involved
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2. Site-specific recombination Occurs between sequences with a limited stretch of similarity ; involves specific sites (recombination sites) Three types/functions: 1. inversion (e.g. expression of alternative genes - inversion of DNA sequence/gene by DNA invertases ) 2. insertion (e.g. infection - insertion of bacteriophages in the bacterial genome by DNA integrase ) 3. deletions (e.g. deletion of bacteriophages from the bacterial genome – different pathway for the bacterium)
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In all three cases the components of the site-directed recombination are: 1.Rearrangement enzymes - recombinases (in some cases accessory proteins are required) Site-specific recombinases cleave and rejoin DNA using a covalent protein-DNA intermediate So, they (have to) have: a) Site-specific endonuclease activity (cleavage) b) DNA ligase activity (rejoining) 2. Recombination site (specific sequence)- places of DNA exchange - specifically recognized by recombinase - partially asymmetric (non-palindromic)
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Site-specific recombination: general Recombination sites in: DNA molecule Rearrangement (enzymes/example) Same orientation Two different DNA molecules Insertion ( integration ; lambda integrase ) Same orientation Same DNA molecule Deletion ( excision ) Inverted orientation Same DNA molecule Inversion ( DNA invertase )
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DNA deletions and insertions Used to recycle genes - recombination sites are recognized by specific recombinase - recombination sites are flanking the gene (e.g. antibiotic resistance gene) in direct orientation and on the same DNA molecule STEPS: - recombination sites align next to each other - recombinase resolves the structure and cuts out (deletes) the gene.
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When the lambda DNA enters the cell, the ends join to form a circular DNA molecule •lambda bacteriophage can multiply in E. coli by a lytic pathway (will destroy the cell), or it can insert its DNA into bacterial genome and enter a latent prophage state . Prophage can exit the host chromosome and shift to lytic growth (pink arrows). Both the insertion of the lambda phage DNA into bacterial genome and its deletion from it, are accomplished by a site-specific recombination event, catalyzed by the lambda integrase enzyme; insertion involves some bacterial enzyme as well. EXAMPLE:
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DNA inversions DNA invertases - recombination sites are repeated sequences but in inverted orientation on the same DNA molecule - recombination sites flank the DNA sequence which is to be inverted STEPS: - recombination sites align next to each other - cross-over and resolution by the recombinase DNA sequence is inverted.
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This note was uploaded on 10/31/2011 for the course BIOL 308 taught by Professor Miskovic during the Winter '09 term at Waterloo.

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308 Lecture 15 - Genetic recombination 1. General or...

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