Site specific recombination

site specific recombination with case studies

1. Site specific Recombination Presented by Roshan Parihar,Ph.D . 1 st yr. Deptt . Of G&PB,COA,IGKV,Raipur

2. Structure involved in CSSR . The pair of symmetric recombinase recognition sequences flank the cross over region where recombination occurs, The subunits of the recombinase binds these recognition sites.notice that the sequence of the crossover regionn is not pallindromec,resulting in an intrinsic assymetry to the recombination sites

3. Tyrosine recombinase family

4. Fig: Recombination by Tyrosine recombinase Here the R1,and R3 ubunits cleave the DNA in first step (A) ,in the example shown ,the protein become linked to the cut DNA by the 3 prime P-tyrosine bond.Exchange of the pair of strands occurs when the two 5 prime OH groups at the break sites each attack the protein DNA bond on the other DNA molecule (B) The second strand excahange occurs by the same mechanism using R2 and R4 sununits

5. Serine recombinase family 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

6. Fig: Covalent-intermediate mechanism used by the serine and tyrosine recombinase Here an OH group from an active site serine is shown to attack the phosphate and there by introduce a single stranded break at the site of recombination. The liberated OH group on the broken DNA can then re-attack the protein-DNA covalent bond toreverse this cleavage reaction,reseal the DNA,Reseal the protein.The recombinase,Labelled Rec,is shown in blue

7. Fig: Recombination by serine recombinase Each of the four DNA strands is cleaved with the cross over region by one subunit of the protein. The subunits are labelled as R1,R2,R3,R4. cleavage othe two individualstrands of one DNA duplex is staggered by the two bases.This two base region forms a hybrid duplex in the recombinant products. The recombination sitesare similar to those shown in figure

8. Figure :Two classes of genetic recombination The top panel shows an example of site specific recombination, the recombination between red and blue sites inverts the DNA segment carrying the A and B genes The bottom panel is the example of transposition in which the red transposable element excise from the grey DNA and inserts into an unrelated site in the blue DNA

9. Fig:Integrtion of the ƛ genomeinto the chromosome of the host cell

10. Three types of CSSR recombination In all the cases red DNA segment is rearranged or moved during recombination ,A,B,X,Y are genes lie in segments of DNA

11. Fig: Mechanism of the site specific recombination by the Cre recombinase

12. Applications Tracking cell lineage during development work was done in Drosophila using the Flp -FRT system Ablating a gene function during development Inducing the expression of a gene at a specific time in development Site-specific recombination in biotechnological applications Targeted mutation in a reverse genetic approach.

13. Biological roles of site specific recombination Phage use CSSR for integration of their genome to host. Use to alter gene expression by inversion CSSR maintaion structural intergity of circular dna molecules cylce of DNA replication,HR,and cell division Recombinases convert the multimeric circular DNA into monomers

14. CASE STUDY FOR KNOCK OUT THE GREY GENES FROM GREY MOUSE FROM WHITE MOUSE White mouse

15. Electroporation used to introduce the DNA HR used the natural mechanism to cross exchange the DNA in mouse GFP of Resistance marker gens is used to select the transformant

16. Transformed cell implanted to grey mouse for fostering