1. Lecture 9 Site Specific Recombination and Transposition Quiz 5 due today at 4 PM

2. 12_Figure01.jpg Examples of site specific recombination and transposition Different than homologous recombination “jumping gene”

3. Responsible for important DNA rearrangements CSSR = recombination between 2 defined sites CSSR can be used to control gene expression. DNA inversion can allow an alternative gene to be expressed. Transposition = recombination between specific sequences and non-specific DNA sites Transposition is a (the) major source of spontaneous mutation. Nearly half the human genome is transposon-derived sequences. Conservative site specific recombination (CSSR) and transposition

4. 12_Figure02.jpg Certain viruses use site specific recombination to integrate into the host chromosome Example: lambda phage

5. 12_Figure03.jpg 3 Types of conservative site specific recombination (CSSR) A, B, X, and Y denote specific genes. The recombinase recognition sequences are dark orange and blue. Gray regions with white arrows are the crossover regions. Direct repeats Direct repeats Inverted repeats 2 sites on different DNA molecules 2 sites on the same DNA molecule

6. 12_Figure04.jpg Structures involved in CSSR Example of an insertion (orange DNA is usually circular) Recombinase recognition sequences are symmetric Crossover region is assymetric 4 subunits of recombinase bind

7. 12_Figure05.jpg Synaptic complex 2 types of recombinases Serine recombinase and tyrosine recombinase Both use a covalent protein-DNA intermediate Conservative in CSSR refers to energy; No ATP needed Recombinase binds 5’- phosphate Leaves free 3’-OH to bind another DNA strand Links DNA to 3’-OH on new strand

8. 12_Figure06.jpg Mechanism of serine recombinase Makes double stranded break that is staggered by 2 bases All 4 strands cleaved before strand exchange 5’- phospho-serine and free 3’-OH formed. R2 segment recombines with R3 segment R4 segment recombines with R1 segment

9. 12_Figure08.jpg Mechanism of tyrosine recombinase Break and rejoin one DNA strand at a time. R1 and R3 break and rejoin segments first. 3’- phospho-tyrosine and free 5’-OH formed. A Holliday junction is formed. R2 and R4 segments then recombine using the same mechanism to resolve junction.

10. 12_Figure09a.jpg Phage P1 Cre recombinase is a tyrosine recombinase sometimes used in genetic engineering

11. 12_Figure15.jpg Homologous recombination during DNA replication of circular chromosomes can generate circular multimers. CSSR is needed to resolve these multimers back into the monomers. These recombinases are called resolvases. XER tyrosine recombinase (resolvase) is an example from bacteria Animation

12. 12_Figure16.jpg Transposable elements or transposons Little selectivity in site selection. Can insert in the middle of genes. Less than 2% of human transposons code for proteins. Non-replicative (cut & paste) Replicative (copy & paste)

13. 12_UnFigure06.jpg

14. 12_Figure17.jpg Some organisms (yeast, fruitflies, and E. Coli) contain few transposons. Occurrence of transposons (green) in genomes

15. 12_Figure18.jpg 3 classes of transposons (LTR retrotransposons) (Long terminal repeat) (Reverse transcriptase) RNA binding enzyme RT & endonuclease/ Rnase H Non-viral retrotransposons

16. 12_Figure19.jpg DNA transposons: Cut & paste mechanism of transposition Old host site is gray New host site is blue Double strand break at old site must be repaired Staggered cleavage of new target site DNA New DNA synthesis gives rise to target site duplication (transpososome)

17. 12_Figure20.jpg DNA strand transfer 3’-OH of transposon attacks phosphate in DNA to leave a new 3’-OH for polymerase to add onto for the gap to be filled. Then ligase seals nick.

18. 12_Figure21.jpg Transposase only cleaves one strand. 3 ways to cleave nontransferred strand (5’-end) before strand transfer A different endonuclease (TnsA) 3’-OH on trnspsn3’-OH on host DNA

19. 12_Figure22.jpg DNA transposons: Replicative “copy & paste” transposition mechanism Transposase nicks DNA to create the 3’-OH at each end for strand transfer (same as in cut & paste) 3’-OH on each end cleaves and binds target DNA DNA replication machinery then uses each 3’-OH on target DNA as a primer and copies the transposon This leaves a large circular DNA molecule containing 2 copies of the transposon Animation

20. 12_Figure23.jpg Virus-like retrotransposons use an RNA intermediate but otherwise use the same mechanism as in DNA transposition Integrated virus Virus uses an integrase related to DNA transposases. Integrase cleaves 2 nucleotides from the 3’-end, which is then used for DNA strand transfer. Viruses need to use a special mechanism to regenerate the ends of the LTRs so they can be recognized by integrase.

21. 12_Figure26.jpg Target-site-primed reverse transcription of Poly-A (non-LTR) retrotransposons Human transposon LINEs (long interspersed nuclear elements) use this mechanism. LINE DNA transcribed. mRNA exported from nucleus. ORF1 and ORF2 proteins are translated from mRNA, remain attached to 3’-end of transcript, and transport mRNA back into the nucleus where it associates with T- rich DNA by use of the poly-A tail To form a DNA:RNA hybrid. ORF2 (RT and endonuclease) nicks DNA generating a 3’-OH for reverse transcription of RNA back into DNA that integrates into host DNA. DNA joining and repair establish the newly produced LINE element.

22. 12_Figure32.jpg Genetic organization of a LINE and SINE 20 % of the human genome is LINE elements (example L1). LINEs donate the proteins to replicate SINEs (100-400 bp sequences that make up 13 % of human genome). The Alu sequence is a SINE. Pseudogenes can be made by LINE proteins mistakingly binding and reverse transcribing normal mRNA.

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