1. Chapter 11 Site-Specific Recombination & Transposition of DNA

2. Two classes of genetic recombination
Conservative site-specific recombination (CSSR)
Transpositional recombination

3. OUTLINE Conservative Site-Specific Recombination
Biological Roles of Site-Specific Recombination
Transposition
Examples of Transposable Elements and Their Regulation
V(D)J Recombination

4. Conservative Site-Specific Recombination(CSSR)
CSSR is recombination between two defined sequence elements

5. 1-1 CSSR occurs at specific DNA sequences in the target DNA
CSSR can generate three different types of DNA rearrangements:
1.Insertion
2.Deletion
3.Inversion

7. Structures involved in CSSR

8. 1-2 Site-specific recombinases cleave and rejoin DNA using a covalent protein-DNA intermediate
There’re two families of conservative site-specific recombinases:
1. Serine Recombinases
2. Tyrosine Recombinases

9. 1-3 Serine recombinases introduce double-stranded breaks in DNA and then swap strands to promote recombination
First , the serine recombinases cleave all four strands
Second, DNA swap occurs
Finally, the serine recombinases are liberated and they seal the DNA strands

10. Recombination by a serine recombinase

11. 1-4 Tyrosine recombinases break and rejoin one pair of DNA strands at a time
In contrast to the serine recombinases ,the tyrosine recombinases cleave and rejoin two DNA strands first, and only then cleave and rejoin the other two strands.

12. Recombination by a tyrosine recombinase

13. 1-5 Structure of tyrosine recombinases bound to DNA reveal the mechanism of DNA exchange
Cre is an enzyme encoded by phage P1 , which functions to circularize the linear phage genome during infection
The recombination sites of Cre on the DNA are called lox sites.
Only Cre protein and the lox sites are needed for complete recombination

14. Biological roles of site-specific recombination
2-1 l integrase promotes the integration and Excision of a Viral Genome into the Host Cell Chromosome

15. Bacteriophage l infects a host bacterium and would establish a lysogen ,which requires the integration of phage DNA into host chromosome
To integrate, lInt catalyzes recombination between two specific sites—attachment (att) sites
attP site is on the phage DNA and attB site is on the bacterial genome
lInt is a tyrosine recombinase, and the mechanism of strand exchange follows the pathway described above for the Cre protein

16. The highly asymmetric organization of the attPand attB sites is important to the regulation of l integration
The following figure showing:
recombination sites involved in l integration and excision showing the important sequence element

18. 2-2 Phage l excision requires a new DNA-binding protein
Phage l excision requires an architectural protein called Xis, which is phage-encoded
Xis binds to the integrated attR sites to stimulate excision and to inhibit integration

19. 2-3 The Hin recombinase inverts a segment of DNA allowing expression of alternative genes
The Salmonella Hin recombinase inverts a segment of the bacterial chromosome to allow expression of two alternative sets of genes
Hin recombinase is an example of programmed rearrangements in bacteria
In the case of Hin inversion,recombination is used to help the bacteria evade the host immune system
Hin is a serine recombinase which promotes inversion

20. 2-4 Hin recombination requires a DNA enhancer
Hin recombination requires a DNA enhancer in addition to the hix sites
Enhancer function requires the bacterial Fis protein
the enhancer-Fis complex activates the catalytic steps of recombination
Hin-catalyzed inversion is not highly regulated, rather, inversion occurs stochastically

21. 2-5 Recombinase converts multimeric circular DNA molecules into monomers
circular DNA molecules sometimes form dimers and even higher multimeric forms during the process of homologous recombination
Site-specific recombinases (sometimes called resolvases) can resolve dimers and larger multimers into monomers

22. Circular DNA molecules can form multimers

23. Xer recombinase is a tyrosine
Xer catalyzes the monomerization of bacterial chromosomes and of many bacterial plasmids
Xer is a heterotetramer, containing two subunits of XerC and two subunits of XerD
XerC and XerD recognize different sequence
The directional regulation of Xer-mediated recombination is achieved through the interaction between the Xer recombinase and a cell diversion protein called FtsK

24. Pathways for Xer-mediated recombination at dif

25. 2-6 There are other mechanisms to direct recombination to specific segaments of DNA
The gene rearrangements responsible for assembly of gene segments encoding critical proteins for the vertebrate immune system—known as V(D)J recombination—also occurs at specific sites

26. Transposition
3-1 Some genetic elements move to new chromosomal locations by transposition
Transposition is a specific form of genetic recombination that moves certain genetic elements from one DNA site to another
These mobile genetic elements are called transposable elements or transposons

27. Transposition of a mobile genetic element to a new site in host DNA

28. The transposons can insert within genes or regulatory sequence of a gene, which results in the completely disruption of gene function
They can also insert within the regulatory sequences of a gene where their presence may lead to shanges in how that gene is expressed
Transposable elements are present in the genomes of all life-forms. (1) transposon-related sequences can make up huge fractions of the genome of an organism. (2) the transposon content in different genomes is highly variable

29. 3-2 There are three principle classes of transposable elements

30. 3-3 · The recombinase responsible for transposition are usually called transposases or ,sometimes,integrases
DNA transposons carry a transposase gene, flanked by recombination sites
DNA transposons carry a gene encoding their own transposase, sometimes they may carry a few additional genes
3-4 · Transposons exist as both autonomous and nonautonomous elements(Autonomous transposons and Nonautonomous transposons)

31. 3-5 · Viral-like retrotransposons and retroviruses carry terminal repeat sequences and two genes important for recombination
Viral-like retrotransposons and retroviruses carry LTRs
Viral-like retrotransposons encode two proteins needed for their mobility: integrase and reverse transcriptase (RT)
3-6 · Poly-A retrotransposons look like genes

32. 3-7 DNA transposition by a cut-and-paste mechanism
The movement of a DNA transposon by a non-replicative mechanism called cut-and-paste transposition
1.First , transposase binds to the terminal inverted repeats at the end of the transopon and brings the two ends of the transopon DNA together to generate a stable protein-DNA complex called the synaptic complex or transpososome

33. 2.Next, the transopon DNA is excised from its original location in the genome
3.Then, the 3’-OH ends of the transopon DNA attack the DNA phosphodiester bonds at the site of the new insertion, this DNA segment is called the target DNA
4.At last, the transopon DNA is covalently joined to the DNA at the target site by DNA strand tranfer. This reaction introduced a nick into the target DNA

34. The cut-and-paste mechanism of transposition

35. The intermediate in cut-and-paste transposition is finished by gap repair
1.Two introduced nick are filled by a DNA repair polymerase(encoded by the host cell) and a DNA ligase
2.Filling in the gap gives rise to the target site duplications that flank transposons

36. There are multiple mechanisms for cleaving the nontransferred strand during DNA transposition
1.An enzyme other than tranposase can be used to cleave the nontransfered strand
2.The ranposase itself cleave the nontransfered strand by using an unusual DNA transesterification mechanism
3.DNA cleavage via a transesterification reaction can also occur between two ends of the transposon

37. 3-8 DNA transposition by a replicative mechanism
First, the transposase protein is assembled on the two ends of the transposon DNA to generate a transpososome
Then, DNA is cleaved at the ends of the transposon DNA

38. Then, the 3’OH ends of the trsnsposon DNA are joined to the target sites by the DNA strand transfer reaction, which generate a doubly branched DNA molecule
At last, The two DNA branches within this intermediate have the structure of a replication fork, and the DNA synthesis is proceeded
This replication reaction generates two copies of the transposon DNA

39. 3-9 Viral-like retrotransposons and retroviruses move using an RNA intermediate
Recombination for retroelements involves an RNA intermediate
A cycle of transposition starts with transcription of the retrotransposon (or retroviral) DNA sequence into RNA by cellular RNA polymerase. Transcription initiates at a promoter sequence within one of the LTRs.
The RNA is then reverse transcribed to generate the cDNA

40. 3. The cDNA is recognized by Integrase and recombinate with a new target DNA site
4. Integrase assembles on the ends of this cDNA and cleaves a few nucleotides off the 3’ ends of each strand
5. Integrase catalyzes the insertion of cleaved 3’ ends into a DNA target site in the host cell genome using the DNA strand transfer reaction.
6. Gap repair reaction generates target-site duplications.

41. Mechanism of retroviral integration and transposition of viral-like retrotransposons

42. 3-10 DNA transposases and retroviral integrases are members of a protein superfamily
Many different tranposases and integrases carry a catalytic domain that has a common three-dimensional shape
This domain contains two D and a E
The tranposase/integrase proteins use this same site to catalyze both the DNA cleavage and the DNA strand transfer
Tranposases and integrases are only active when assembled into a synaptic complex, also called a transpososome, on DNA

43. 3-10 Poly-A Retrotransposition move by a “reverse splicing” mechanism
The Poly-A Retrotransposons use an RNA intermediate but use a mechanism different than that used by the viral-like elements. This mechanism is called target site primed reverse transcription
1. First, the DNA of an integrated element is transcripted by a cellular RNA polymerase
2. Then, newly synthesized RNA is exported to cytoplasm to produce ORF1 and ORF2 proteins

44. 3. The protein-RNA complex then reenters the nuclease and associates with the cellular DNA
4.The endonuclease initiates the intergration reaction by introducing a nick in the chromosomal DNA
5. The 3’OH DNA end generated by the nicking action then serves as the primer for reverse transcription of the element RNA

45. Transposition of a poly-A retrotransposon by target site-primed reverse transcription

47. Examples of transposable enements and their regulation
Two types of regulation appear as recurring themes:
Trnasposons control the number of their copies present in a given cell
Trnasposons control target site choice

48. 4-1 IS4-family transposons are compact elements with multiple mechanisms for copy number control
Tn10 transposes via the cut-and paste mechanism, using the DNA hairpin strategy to cleave the nontransfered strands
Tn10 limits its copy number in any given cell by strategies that restrict its transposition frequency. One mechanism is the use of an antisense RNA to control the expression of the transposase gene
By this mechanism, cells that carry more copes of Tn10 will transcribe more of the antisense RNA, which in turn will limit expression of the transposase gene. The transposition frequencywill,therefore, be very low in such a strain

49. Antisense regulation of Tn10 expression

50. 4-2 Transposition is coupled to cellular DNA replication
Bacteria methylate their DNA at GATC sites and GATC sites are hemimethylated for a few minutes
It is during the brief period—when the Tn10 DNA is hemimethylated—that transposition is more likely to occur
Both RNA polymerase and transposase bind more tightly to the hemimethylated sequences than to their fully methylated versions. As a result, when the DNA is hemimethylated, the transposase gene is most efficiently expressed, and the transposaseprotein binds most efficiently to the DNA

51. Transposition of Tn10 after passage of a replication fork

52. V(D)J recombination
The principal mechanism cells use to generate antibodies and T cell receptors with such diversity relies on a specialized set of DNA rearrangement reactions known as V(D)J recombination

53. Overview of the process of V(D)J recombination

54. The early events in V(D)J recombination occur by a mechanism similar to transposon excision
Revombination sequences, called recombination signal sequences, flank the gene segments that are assembled by V(D)J recombination
Recombination always occurs between a pair of recombination signal sequences which are organized as inverted repeats flanking the DNA segments that are destined to be joined

55. First, reconbinase recognizes the recombination signal sequences and pairs the two sites to form a protein-DNA synatic complex
Then, the RAG1 proteins within this complex introduce single-stranded breaks in the DNA at each of the junctions between the recombination signal sequence and the gene segment that will be rearranged
Then, this 3’OH DNA end attacks the opposite strand of the DNA double helix,which results in the generation of a hairpin DNA end

56. The V(D)J recombination pathway

57. THE END
THANK YOU!