Site specific recombination
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Site-specific recombination involves DNA strand exchange between segments with sequence homology, mediated by site-specific recombinases (SSRs). SSRs recognize and bind to short DNA sequences, cleaving the DNA backbone to exchange helices and rejoin strands. They are classified into tyrosine and serine recombinase families based on mechanism. Tyrosine recombinases like Cre and Flp cleave DNA strands staggered by 6-8bp, linking DNA ends to the recombinase. Serine recombinases simultaneously cleave all four strands staggered by 2bp via phosphoserine bonds. SSRs have applications like tracking cell lineage, ablating genes, and inducing gene expression at specific developmental times.
Site specific recombination
- 2. • conservative site-specific recombination, • is a type of genetic recombination in which DNA strand exchange takes place between segments possessing at least a certain degree of sequence homology. • Bacteriophage genome into bacterial chromosome • Site-specific recombinases (SSRs) perform rearrangements of DNA segments by recognizing and binding to short DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA helices involved and rejoin the DNA strands.
- 4. • They are employed in a variety of cellular processes, including bacterial genome replication, differentiation and pathogenesis, and movement of mobile genetic elements
- 5. • Recombination sites are typically between 30 and 200 nucleotides in length and consist of two motifs with a partial inverted-repeat symmetry, to which the recombinase binds, and which flank a central crossover sequence at which the recombination takes place. The pairs of sites between which the recombination occurs are usually identical, but there are exceptions
- 7. Classification • Based on amino acid sequence homology and mechanistic relatedness most site-specific recombinase are grouped into one of two families: 1. tyrosine recombinase family 2. Serine recombinase family members of the serine recombinase family were known as resolvase / DNA invertases • member of the tyrosine recombinases, lambda- integrase, using attP/B recognition sites
- 9. • During strand exchange, the DNA cut at fixed points within the crossover region of the site releases a deoxyribose hydroxyl group • recombinase protein forms a transient covalent bond to a DNA backbone phosphate. • This phosphodiester bond between the hydroxyl group of the nucleophilic serine or tyrosine residue conserves the energy that was expended in cleaving the DNA. • Energy stored in this bond is subsequently used for the rejoining of the DNA to the corresponding deoxyribose hydroxyl group on the other site.
- 10. • Tyrosine recombinases, such as Cre or Flp, cleave one DNA strand at a time at points that are staggered by 6-8bp, linking the 3’ end of the strand to the hydroxyl group of the tyrosine nucleophile. • Strand exchange then proceeds via a crossed strand intermediate analogous to the Holliday junction in which only one pair of strands has been exchanged
- 13. • of serine recombinases is much less well understood. • classical members are gamma-delta and Tn3 resolvase, but also new additions like φC31-, Bxb1-, and R4 integrases, cut all four DNA strands simultaneously at points that are staggered by 2bp . • During cleavage, a protein-DNA bond is formed via a transesterification reaction in which a phosphodiester bond is replaced by a phosphoserine bond between a 5’ phosphate at the cleavage site and the hydroxyl group of the conserved serine residue
- 15. Applications • Tracking cell lineage during development work was done in Drosophila using the Flp- FRT system
- 16. • Ablating a gene function during development
- 17. • Inducing the expression of a gene at a specific time in development
- 18. • Site-specific recombination in biotechnological applications