Chapter11

1. Chapter11 Site-specific Recombination and Transposition of DNA 生物学基地班 200431060054 郑丽娟

2. The subject of this chapter those progress that rearrange DNA sequences and thus lead to a more dynamic genome structure

3. There are two classes of genetic Recombination thatare responsible for many important DNA rearrangements: conservative site-specific Recombination (CSSR) transpositional recombination (transposition)

4. Site-specific Recombination Occurs at Specific DNA Sequences in the Target DNA • Each recombination site is organized as a pair of recombinase recongnition sequences and the crossover region

5. There are three types of CSSR recombination

6. Site-specific Recombinases Cleave and Rejoin DNA Using a Covalent Protein-DNAIntermediate • Serine recombinases • Thyrosine recombinases

7. Serine recombinases introduce double-stranded breaks om DNA and the swap strands to promote recombination

8. Thyrosine recombinases break and rejoin one pair of DNA strands at a time

9. Structure of tytosine recombinases bound to DNA reveal the mechanism of 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 combination sites on the DNA ,where Cre acts,are called lox sites. Only Cre protein and the los sites are needed for complete recombination

10. Bilogical roles of site-specific recombination • many phage insert their DNA into the host chromosome during infection • Alter fene expression • of DNA replication,homologous Widelu used to help maintain the structural integrity of circular DNA molecules during cycles recombination and cell division

11. The 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

12. l integrase promotes the integration and Excision of a Viral Genome into the Host Cell Chromosome

13. The l integrase protein(lInt)catalyzes recombination between two specfic sites(att sites) attB site in the bacterial chromosome attP site in the phage DNA • integrase is a tyrosine recombinase • Intergration requires accessory proteins IHF is a sequence-dependent DNA binding protein that introduces large bends in DNA Phage excision requires a new DNA-bending protein------Xis

14. The Hin recombinase inverts a segment of DNA allowing expression of alternative genes

15. The Hin recombinase inverts a segmentof the bacterial chtomosome to allow expression of two alternative sets of genes • The genes that are controled by the invertion encond a kind of the protein which is the target for the immune system.So some individuals can avoid recongnition of the immune system

16. Recombinase converts multimeric circular DNA molecules into monomers The chromosomes of most bacteria, plasmids and some viral genomes are circular. These circular DNA sometimes form dimers and even multimeric forms during the process of homologous recombination. Site-specific recombinases can be used to convert these DNA multimers back into monomer

18. The Xer recombinase • It is 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. Xer is a heterotetramer containing two subunits of XerC and two subunits of XerD. XerC and XerD recognize different DNA sequence.

20. TRANSPOSITION

21. 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.

22. 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

24. Viral-like retrotransposons and retroviruses move using an RNAintermediate

25. Viral-like retrotransposons and retroviruses catty ternima repeat sequences and two genes important for recombination Poly-A retrotransposons look like genes ,the two ends of the element have distinct sequences

26. Poly-A Retrotransposons move by a reberrse splicing mechanism • Poly-A Retrotransposons move using an RNA intermediate but use a mechanism different ,which is called terrget site primed reverse transcription • Transcription of the integrated DNA • The newly synthesized RNA is exported to cytoplasm to produce ORF1 and ORF2 proteins, which remain tobind the RNA

27. 3. The protein-RNA complex reenters the nuclease and associate with the chromosomal DNA 4. The endonuclease activity of ORF2 introduce a nick on the chromosomal DNA at the T-rich sites. 5. The 3’OH generated on the target DNA serves as the primer for reverse transcription of the element RNA (ORF2)

28. Cut-and-paste transposition mechnism

30. Transpososome formation

31. Excision of the transposon

34. DNA strand transfer

36. Gap repair

39. The similarity and difference between Replicative Transposition and Cut-and-paste transposition

43. The transposon DNA is duplicated during transposition,leading to two copies of the transposon joined to the new and old DNA target sites

49. Replicative transpisition Mechanism