site specific recombination with case studies

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

6. 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

10. Tyrosine recombinase family

12. 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

13. 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

14. 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

16. 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

19. 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

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

21. 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

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

27. 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.

28. 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

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

30. 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

31. Transformed cell
implanted to grey
mouse for
fostering