Site specific recombination and transposition

1. SITE SPECIFIC
RECOMBINATION
AND TRANSPOSITION
MOLECULAR BIOLOGY
Submitted to:
Dr. Nishita I.K.
Assistant Professor
Department Of Botany
St. Teresa’s College
Submitted by:
Ananya J.S.
Roll No: 5
Department Of Botany
St. Teresa’s College

2. • Two classes of genetic recombination;
1. Conservative site-specific recombination
2. Transpositionalrecombination
2

3. Site Specific Recombination
• It is a type of genetic recombination in which DNA strand exchange
takes place between segments possessing at least a certain degree
of sequence homology.
• It has short sequence elements, called recombination sites, where
the recombination occurs.
• Example; integration of the bacteriophage into bacterial
chromosome.
3

4. 4

5. 3 Different Types of DNA Arrangement
(1). Insertion (2). Deletion (3). Inversion
5

6. Site Specific Recombinases
• Enzymes required for site specific recombination.
• They perform rearrangements of DNA segments by recognizing
and binding to short, specific sequences (sites)
6

7. Structure Involved In CSSR
• The pair of symmetric recombinase
recognition sequences flank the
crossover region where recombination
occurs.
• Subunits of recombinase bind these
sites.
• It notice the sequence of the crossover
region is not palindromic, resulting in an
intrinsic asymmetry to the
recombination sites
7

8. Classification
• Based on amino acid sequence homology and mechanism most site-
specific recombinase are grouped into one of two families;
1. Serine recombinase family
2. Tyrosine 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
8

9. 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
9

10. • 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 of the two
individual strands 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
10

11. Tyrosine Recombinase Family
• 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.
11

12. • Here the R1,and R3 subunits 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 exchange occurs by
the same mechanism using R2 and R4
subunits
12

13. 13

14. 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.
14

15. Biological Roles
• Phage use CSSR for integration of their genome to host.
• Use to alter gene expression by inversion.
• CSSR maintain structural integrity of circular DNA molecules cycle of
DNA replication, HR, and cell division.
• Recombinases convert the multimeric circular DNA into monomers.
15

16. Transposition
• The transpositions is the movement of genetic element called
transposons or transposable element between plasmids and
genomes.
• TRANSPOSONS: A DNA sequence able to insert itself (or a copy of
itself) at a new location in the genome without having any
sequence relationship with the target locus
16

17. Transposable Elements
• A sequence of DNA that can change its position within a genome.
Also known as Transposable Elements(TE).
• Transposition often results in the duplication of the transposable
elements.
• Popularly called jumping genes.
• Discovered by Barbara McClintock in 1940.
• Won the Nobel Prize in 1983 for the same.
17

18. Transposons
• They are repetitive DNA sequences that have the capacity to move
from one location to another in genome.
• Transposon movement can result in mutations, alter gene expression,
induce chromosome rearrangements and, due to increase in copy
numbers, enlarge genome sizes.
• They are powerful forces of genetic change and have played a
significant role in the evolution of many genomes.
• DNA transposons can be used to introduce a piece of foreign DNA into
a genome
18

19. 3 Classes Of Transposons
• Class I transposons – Retrotransposons; an elements’ RNA is reverse
transcribed using reverse transcriptase into DNA molecules and these
DNA molecules are subsequently inserted into new genomic locations.
• Class II transposons; DNA segments move via a “cut and paste”
mechanism in which the transposon is excised from one location and
reintegrated elsewhere. DNA transposons consist of a transposase gene
that is flanked by two Terminal Inverted Repeats. The transposase
enzyme recognizes these inverted repeats to perform the excision and
then the DNA segment is inserted into a new genomic location.
19

20. • Class III transposons
– Miniature
inverted-repeat
transposable
elements; an
element is replicated
and one copy is
inserted at a new
site, while the other
is retained at the
original site. Eg: Tn3
element
20

21. 21

22. Retrotransposons
• Retrotransposons – also called transposons via RNA intermediate.
• They can amplify themselves in a genome and are ubiquitous
components of the DNA of eukaryotes.
• These DNA sequences use a ‘copy-and-paste’ mechanism.
22

23. 23

24. Retrotransposons – Biological Activity
• Rapidly increase the copy numbers of elements, thereby increasing
genome size.
• Induce relatively stable mutations by inserting near or within genes.
• Transposition and survival of retrotransposons within the host
genome are regulated both by the retrotransposon and host-
encoded factors.
• Because of accumulated mutations, most retrotransposons are no
longer able to retrotranspose.
24

25. Uses Of Transposons
• As cloning vehicles
• A Transformation vectors for transferring genes between organisms.
• Also drug resistance genes encoded by many transposons are useful
in the development of plasmids as cloning vehicles.
• Transposons mutagenesis
• Use of transposons to increase rate of mutation due to insertional
inactivation
25

26. Mechanism
• The insertion of a transposon into a new site
• It consist of making staggered breaks in the target DNA, joining the
transposon to the single stranded ends, and filling in the gaps.
• The generation and filling of the staggered ends explains the
occurrence of the direct repeats of target DNA at the site of insertion.
• The stagger between the cuts on the two strands determines the
length of the direct repeats; thus the target repeat characteristic of
each transposon reflect the geometry of the enzyme involved in
cutting target DNA.
26

27. Classification
• The use of staggered ends is common to most means of
transposition, but we can distinguish two major types of
mechanisms by which a transposon moves;
1. Replicative transposition
2. Cut and paste transposition
27

28. Replicative Transposition
• In replicativetransposition, the element is duplicated during the reaction
so that the transposing entity is a copy of the original element.
• The transposon is copied as part of its movement.
• One copy remains at the new site, whereas the other inserts at the new
site.
• Thus transposition is accompanied by an increase in the number of
copies of the transposons.
28

29. • Replicative transposition involves two types of enzymatic activity:
1. Transposase
2. Resolvase
• A transposase act on the end of the original transposons
• Resolvase act on the duplicated copies
• While one group of transposons moves only by replicative
transpositions, true replicative transposition is relatively rare
among transposons in general
29

30. • The transposome introducesa single-strand
nick at each of the ends of the transposon DNA.
This cleavage generates a 3’OH group at each
end. These OH groups then attack the target
DNA and become joined to the target by DNA
strand transfer.Note that at each end of the
transposon,only one strand is transferredinto
the target at this point, resultingin the
formation of a doubly-branchedDNA structure.
• The replication apparatus assembles at one of
these “forks” (the left one in the figure).
Replication continuesthrough the transposon
sequence.The resultingproduct,called a
cointegrate,has the two startingcircular DNA
molecules joined by two copies of the
transposon.The ssDNA gaps in the branched
intermediategive rise to the target site
duplications.These duplicationsare not shown
in the cointegratefor clarity.
30

31. Cut And Paste Transposition
• In non replicative transposition
• This recombination pathway involves the excision of the transposon
from its initial location in the host DNA followed by integration of this
excised transposon into a new DNA site.
• To initiate recombination, the transposase binds to the terminal inverted
repeats at the end of the transposon.
• It brings the two ends of the transposon DNA together to generate a
stable protein-DNA complex, known as synaptic complex or
transpososome.
31

32. • This complex functions to ensure that the DNA cleavage and joining
reactions needed to move the transposon occur simultaneously on the
two ends of the element’s DNA.
• It also protects the DNA ends from cellular enzymes during
recombination.
• Transposase cleaves one DNA strand at each end at junction between
transposon DNA and host DNA, transposon sequence terminates with
free 3’-OH groups at each end.
• Other DNA strands cut by various mechanisms and transposon excised.
32

33. • 3’-OH ends of transposon DNA attack DNA phosphodiester bonds at site
of target DNA.
• Nicks introduced in other target DNA strands few nucleotides apart →
transposon joined via reaction called DNA strand transfer
• Few nucleotides between nicks leaves small ss gaps filled in by host DNA
repair polymerase → small target site duplications on either side
transposon
• DNA ligase seals final nicks
• Ds break where transposon left repaired by homologous recombination.
33

34. • The figure shows movement of a
transposon from a target site in the gray
host DNA to a new site in the blue DNA.
Note the staggered cleavage sites on the
target DNA during the DNA strand
transfer reaction that give rise to short
repeated sequences at the new target
site (the target site duplications). The
DNA at the original insertion site (here in
gray) will be left with a double- stranded
DNA break as a result of transposon
excision. This break can be repaired by
nonhomologous end joining or
homologous recombination
Duplication of target
DNA completed
34

35. Conclusion
• Transposons are present in the genomes of all organisms, where
they can constitute a huge fraction of the total DNA sequence.
They are a major cause of mutations and genome rearrangement.
• The ability of transposable elements to insert and to generate
deletions and inversions accounts for much of the
macromolecular rearrangement.
• They cause mutation which is used in the production of different
colors of grapes, corn and other fruits.
• As a result they are used in the genetic studies.
35

36. Reference
• Peter Snustad,D (2015).PrinciplesOf Genetics.John Wiley & Sons, Inc.
• James D. Watson (2004).Molecular Biology Of The Gene. Pearson Education,Inc.
• https://en.m.wikipedia.org/wiki/Site-specific_recombination
• https://academic.oup.com/femsre/article/21/2/157/594254
• https://www.britannica.com/science/transposon
36

37. THANK YOU