Homologous recombination and Site specific recombination

1. Homologous recombination and Site specific recombination
Presented ,
Mr-Pradeep D Devkate
M.Sc Microbiology, B.Ed, MH-SET, 2-GATE(Biochemistry, Zoology,
Botany)

2. Genome changes
Mutation Recombination
Sudden change in DNA Sequence
Mutagenic agents- Physical, chemical, Biological
Repair system work - Remove the Damage
and add the correct Nucleotides
Genomic Information is Stable.
Minimum changes due to Mutation
Rearrangement of DNA Molecules that involves the
Breakage and reunion of DNA
Minimum Homology required  sharing information
between two strands
Due to recombination progeny differ from his parents

3. Recombination:
The process of exchange of DNA segments is known as Recombination
1) Homologous recombination
 Similar or identical strands.
 Results in the exchange of DNA segments.
Single stranded break = one holliday junction
DS break =two holliday junction
2) Site specific recombination –
 Only a small site is similar in two DNA
 Smaller DNA is integrated in to larger DNA
1)single strand break = tyrosine recombinase
2)double stranded break =serine recombinase
Between Two DNA Molecules that have only short
regions of homology  Site specific recombination
Bacterial DNA phage
Exchange of homologous segments between two
homologous DNA molecules = H.R.

4. Transposons – Not required Homology
• Illegitimate Recombination--- Homology Independent
• Sequence of DNA inserted into Another regions without relying
sequence homology
• Self DNA Code Transposase Enzyme…. Cut to same DNA seq. and paste
to another site,,,,.

5. Homologous recombination:
It also involved in DNA repair.
 Homologous recombination / general recombination
Also occur in all organisms but first discovered in bacterial system
Prokaryotic= conjugation, transduction, Transformation
Eukaryotic= In meiotic phase
Crossing over : reciprocal exchange of DNA segments

6. Holliday model for H. R. / heteroduplex model
Recombination between two homologous DS DNA molecules those
with identical or nearly identical sequences.
Ligation strand produces a crossed strand intermediate called as
Holliday junction
Junction moves / Branch migration exchange of longer segments of
DNA
Separation or Resolution of Holliday junction  Cleavage across the
Branch Point

7. Step 1= Pairing of homologous DNA-
Step 2 = single strand break-( by Rec BCD)
Repair of UV Damage(RUV Protein)

8. Step 3- strand invasion (by Rec A)
Step 4- Branch migration (Ruv AB)
Holliday junction 
Crossover hotspot

9. Step 5- resolution (Ruv C)
Vertical cut = all recombinant
Horizontal cut= all parental

11. 2) Site specific recombination or
conserved site specific recombination (CSSR)
• In Site specific recombination few nucleotide are similar between two
DNA .
• A specific site which are present called LOX-P Site.
att-B att-P
Integrase
att-L=BOP
att-R= POB
Sequence B And P are common in Both Phage and Bacteria is= O
After crossing over this attachment side
become hybrid ,,, and now they are known as
attachment-L and att-R ..

12. att-B att-P
Integrase
att-L=BOP
att-R= POB
o Lambda phage ds-DNA incorporated into Bacterial DNA called prophage.
o O is identical centre region of both= Short Homologous Region
o Tyrosine recombinase  Break phosphodiester Bond and covalently attaches to 3’-
Phosphate group

13. Cre-Lox System
 Cre = Causes Recombination (Recombinase Enzyme) – Tyrosine Recombinase
 Lox P : Locus of X-gene Over P
 Cre-Recombinase Recognizes 34 bp site in DNA called Lox-P site.
 Cre-Recombinase catalysis Recombination between two Lox-P site.
 It is a site specific recombination technology which is used to carry out
deletion, insertion , and translocation and inversion
 it can used in eukaryotic and prokaryotic

14. Case 1- two Lox-P site are present in two
different DNA
• Result = Reciprocal translocation

15. Case-2 Two Lox-P site are present in same
DNA and Same Direction
• Deletion gene

16. Case-3 Two Lox – P-sites in same DNA and in
opposite direction

18. • Lox-P site –same direction—Deletion of gene
• Lox –P site opposite site ---- Inversion of Gene
• Lox-P site on different chromosome--- Reciprocal Translocation

19. Two strands are 99 % similar , in this strands
which type of recombination is possible ???

20. Proteins Involved in Homologous Recombination in Prokaryotic
Rec B,C,D – processing of ds DNA break.(Helicase , Nuclease Activity)Exonuclease-V
step-2= Rec-D 5’3’ exonuclease (ATP Dep)
Rec-B 3’5’ Exonuclease (ATP Dep)
Activity of enzyme is altered when it interact with Chi sequence(5’GCTGGTGG3’) in E.Coli.
RecA first binds cooperatively to the invading single strand, help to pairing of homologous strands.
Rec-C Recognized Chi Sequence
Rec-C Signal to Rec-D to stop Unwinding of DNA
 RUV-A,B – Branch Migration steps-10-20 bp/sec
 RUV-A  specific DNA Binding Protein , Recognize and Bind to Holliday junction and Recruits
RUV-B Protein
 RUV-B ATPase Activity provide energy for Branch Migration
 RUV-C – Resolution ( Holiday Junction)
 cleavage  Resolves of the holliday junction
Unwinding
1.Rec A,B,C= Recombination Protein A,B,C.. 2.RuvABC (Recombination UV)

21. ??????????????????
Thank You….
• For any query or suggestion please mail at:-
pradeep.devkate@gmail.com