Site-Specific Recombination & Transposition of DNA

1. Chapter 11 Site-Specific Recombination & Transposition of DNA 生物科学类 张晓娇 200431060149

2. Introduce The subject of this chapter: genetic processes that rearrange DNA sequences and thus lead to a more Dyn-amic genome structure

3. Two classes of genetic recombi-nation • Conservative site-specific recombi-nation (CSSR):

4. Transpositional recombination

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

6. Conservative Site-Specific Recombination

7. Conservative Site-Specific Recombination a . Site-specific Recombination Occurs at Specific DNA Sequences in the Target DNA • Recombination sites (where DNA exchange occurs):the segment of DNA that will be moved carries specific short sequence elements .

8. Conservative Site-Specific Recombination an example: λ integration

9. Conservative Site-Specific Recombination The three types of CSSR recombination Depends on the organization of the recombination sites on the DNA molecule or molecule that participate in recombination.

10. Conservative Site-Specific Recombination Structures involved in CSSR

11. Conservative Site-Specific Recombination b. Site-specific recombinases cleave and rejoin DNA using a covalent protein-DNA intermediate • Serine recombinases • Thyrosine recombinases

12. Conservative Site-Specific Recombination Covalent-intermediate mechanism used by the serine and tyrosine

13. Conservative Site-Specific Recombination c. Serine recombinases introduce double-stranded breaks in DNA and then swap strands to promote recombination • The serine recombinases cleave all for strands prior to strand exchange. These double-stranded DNA breaks in the parental DNA generate four double-stranded DNA segments.

14. Conservative Site-Specific Recombination Recombination By A serine recombinase

15. Conservative Site-Specific Recombination d. 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 stands.

16. Conservative Site-Specific Recombination Recombination By A tyrosine Recombinase

17. Conservative Site-Specific Recombination e. Structure of tyrosine recombinases bound to DNA reveal the mechanism of DNA exchange • Cre is an phage P1-encoded protein, functioning to circularize the linear phage genome during infection • Cre is a tyrosine recombinase • The recombination sites of Cre is lox sites. Cre-lox is sufficient for recombination

18. Conservative Site-Specific Recombination Cre recombinase

19. Conservative Site-Specific Recombination Mechanism of site-specific recombination by The Cre recombinase

20. Conservative Site-Specific Recombination

21. Biological Roles of Site-Specific Recombination

22. Biological Roles of Site-Specific Recombination Some functions of site-specific recombination • Many phage insert their DNA into the host chromosome during infection using this recombination mechanism • Alter gene expression. ＊inversion of a DNA segment can allow two alternative genes to be expressed • Help Maintain the structural integrity of circular DNA molecules during cycles of DNA replication, homologous, recombination, and cell division

23. Biological Roles of Site-Specific Recombination Some general themes of site-specific recombination • All reactions depend critically on the assembly of the recombinase protein on the DNA, and the bringing together of the two recombination sites • For some recombination this assembly requires only the recombinase and its recognition sequence ,others requires accessory proteins including Architectural Proteins

24. Biological Roles of Site-Specific Recombination a. l integrase promotes the integration and Excision of a Viral Genome into the Host Cell Chromosome • To integrate, the l integrase protein(l lnt) catalyzes recombination between two specific sites, known as the att, or attachment, sites. The attP site is on the phage DNA, and the attB site is in the bacterial chromosome. P for phage. B for bacteria.

25. Biological Roles of Site-Specific Recombination Important to the regulation of l integtation is the highly asymmetric organization of the attP and attB sites

26. Biological Roles of Site-Specific Recombination b. Phage l excision requires a new DNA-bending protein • l excise an additional architectural protein, this one phage-encoded is essential for excisive recombination. • This protein Called Xis, binds to specific DNA sequences and introduces bends in the DNA. its dual action as a stimulatory cofactor for excision and an inhibitor of integration ensures that the phage genome will be free, and remain free, from the host chromosome when Xis is present.

27. Biological Roles of Site-Specific Recombination c. 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. • An example of a class of recombination reactions common in bacteria. • known as programmed rearrangements Hin inversion is used to help the bacteria evade the host immune system.

28. Biological Roles of Site-Specific Recombination The genes controlled by Hin inversion encode two alternative forms of flagellin, the protein component of the flegellar fillament. Flagella are on the surface of the bacteria and thus a common target for the immune system Salmonella (showing flegella) invading cultured human cells.

29. Biological Roles of Site-Specific Recombination The chromosomal region inverted by Hin is about 1,000 bp and is flanked by specific recombination sites called hixL and hixR.

30. Biological Roles of Site-Specific Recombination d. Hin recombination requires a DNA enhancer • Hin recombination requires a sequence in addition to the hix sites. This short (~60 bp) sequence is an enhancer that stimulates the rate of recombination ~1,000-fold. • An example of enhancer sequences that stimulate transcription .

31. Biological Roles of Site-Specific Recombination e. Recombinases convert multimeric circular DNA molecules into monomers • The chromosomes of most bacteria, plasmids and some viral genomes are circular. • During the process of homologous recombination, these circular DNA sometimes form dimers and even multimeric forms, which can be can be converted back into monomer by site specific recombination. • Site-specific recombinases also called resolvases catalyze such a process.

32. Biological Roles of Site-Specific Recombination

33. Biological Roles of Site-Specific Recombination Essential that the enzyme catalyze resolution but not the reverse reaction The Xer recombinase is one of these enzymes , Xer recombinase is a tyrosine recombinase and catalyzes the monomerization of bacterial chromosomes and of many bacterial plasmids. a heterotetramer containing two subunits of XerC and two subunits of XerD. XerC and XerD recognize different DNA sequence.

34. Biological Roles of Site-Specific Recombination Pathways for Xer-mediated Recombination At Dif.

35. Biological Roles of Site-Specific Recombination f. There are other mechanisms to direct recombination to specific segments of DNA • Mating type switching in yeast.

36. Transposition Transposition

37. Transposition 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. • Movement occurs through recombination between the DNA sequences at the ends of the transposons and a sequence in the host DNA with little sequence selectivity.

38. Transposition transposons

39. Transposition a. Some genetic elements move to new chromosomal locations by transposition

40. Transposition Transposition of a mobile genetic element to a new site in host DNA,which occurs with or without duplication of the element.

41. Transposition b.There are three principle classes of transposable elements • DNA transposons • Viral-like retrotransposons including the retrovirus, which are also called LTR retrotransposons • Poly-A retrotransposons, also called nonviral retrotransposons

42. Transposition

43. Transposition c. DNA transposons carry a transposase gene, flanked by recombination sites • Recombination sites are at the two ends of the transposon and are inverted repeated sequences varying in length from 25 to a few hundred bp. • The recombinase responsible for transposition are usually called transposases or integrases. • Sometimes they carry a few additional genes. Example, many bacterial DNA transposons carry antibiotic resistance gene.

44. Transposition d. Transposons exist as both autonomous and nonautonomous elements • Autonomous transposons: carry a pair of terminal inverted repeats and a transposase gene; function independently • Nonautonomous transposons: carry only the terminal inverted repeats; need the transposase encoded by autonomous transposons to enable transposition

45. Transposition e.Viral-like retrotransposons and retroviruses carry terminal repeat sequences and two genes important for recombination • Inverted terminal repeat sequences for recombinase binding are embedded within long terminal repeats (LTRs), being organized on the two ends of the elements as direct repeats. • reverse transcriptase (RT), using an RNA template to synthesize DNA. • integrase (the transposase)

46. Transposition

47. Transposition f.Poly-A retrotransposons look like genes • Do not have the terminal inverted repeats. • On end is called 5’ UTR (untranslated region), the other end is 3’ UTR followed by a stretch of A-T base pairs called the poly-A sequence. Flanked by short target site duplication. • Carry two genes. ORF1 encodes an RNA-binding proteins. ORF2 encodes a protein with both reverse transcriptase (RT) and endonuclease activity

48. Transposition g. DNA transposition by a cut-and-paste mechanism

49. Transposition h. DNA transposition by a replicative mechanism/replicative transposition

50. Transposition