1. Picture taken in 1929 of Emerson’s corn cytogenetics class at Cornell University - Beadle is a graduate student shown here with the dog.

2. CHAPTER 29 The Molecular Mechanism of Recombination All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Sections 29.2 and 29.3 pages 953 to 967 Yes, you will see phage experiments AGAIN in this lecture.

3. Genetic Recombination All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 3 different types: 1) Homologous recombination requires homologous sequences get long regions exchanged between homologous sequences 2) Site-specific recombination requires a SPECIFIC short DNA sequence and a recombinase (ex. Viral genome integration) 3) Transposition (“jumping genes”) short sequences (transposons) can excise and reinsert at a different place in the genome.

4. Homologous Recombination (“General recombination”) All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 In bacteria: When new DNA gets into a cell: –by transformation: uptake of naked DNA into a bacterial cell (i.e. what happened in Griffith’s experiment when live R bacteria took up dead S bacterial DNA and were transformed) –by conjugation: chromosome transfer (Hfr strains)

5. Genetic Information Can Be Transferred Between Bacteria In 1946, Lederberg and Tatum showed that two different strains of bacteria with different growth requirements could exchange genes Lederberg and Tatum surmised that the bacterial cells must interact with each other - the process is now known as sexual conjugation

6. Progeny cells had a combination of genetic info from both parents i.e. recombination had occurred

7. Parents must have “interacted” Bacteria Sexual conjugation Fertility factor Has Fertility (F) factor = a plasmid -small DNA circle -extrachromosomal -replicates autonomously Transfers F plasmid to F- cell Via a temporary bridge called a “pilus” -genes for pilus formation are on the F factor plasmid F+ (donates DNA) F- (receives DNA)

8. Bacterial Conjugation F- F+ Pilus

9. 1) Transfer is initiated by a “nick” Single-stranded break in F factor 2) 5’ end is transferred Through pilus to the F- cell 3) Entering F plasmid Is copied 4) conjugation converts the F- cell into an F+ cell

10. F factors can integrate into the host chromosome If an ‘F factor’ integrates it turns the host chromosome into an “Hfr chromosome” and the cell into an Hfr cell Hfr = “high frequency of recombination” Hfr cells can make pili and conjugate with F- cells When the F factor is transferred to F - cells adjacent genes from the chromosome are also transferred

11. Hfr cells can transfer host chromosomal genes Chromosome Transfer in Bacteria From Fig 29.7 The F factor sequence is indicated by the triangle) Genes are transferred in a fixed order, theoretically the whole chromosome can be transferred

12. Homologous Recombination (“General recombination”) All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 In bacteria: When new DNA gets into a cell: – by conjugation: chromosome transfer (Hfr strains) – by transformation: uptake of naked DNA into a bacterial cell During DNA repair: –Probably the most important role of recombination in bacteria – bacterial mutants with a non-functional recombination system have trouble coping with DNA damage

13. Homologous Recombination All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 In eukaryotes: Recombination maintains genetic diversity in a population occurs during meiosis – when diploid germline cells divide to produce haploid gametes (ova and sperm) during DNA repair

14. DNA is the genetic material Meiosis Diploid germ line cell DNA replication Exchange of genetic material 2nd cell division without DNA replication 4 haploid gametes “Mixing” goes on! Recombination

15. Pictures of homologous recombination during meiosis

16. First Mechanistic Clues In 1961, Meselson and Weigle showed: 1) homologous recombination involves the breaking and rejoining of chromosomes (DNA replication is not required) 2) can get recombination products that are “heteroduplexes”

17. Meselson and Weigle Did an experiment with differentially-labeled bacteriophage (viruses that infect bacteria) Used density labels instead of radioactivity 1) Prepared “heavy” phage: labeled with 13 C and 15 N 2) Prepared “light phage”: labeled with 12 C and 14 N 3) Mix both types of phage in one flask with bacteria (under conditions which inhibit DNA replication) injected viral DNA gets packaged into new particles 4) separate viral progeny on a density gradient Q: Do you get intermediate density viruses?

18. Meselson & Weigle - part 1 Note the recombined viral genomes

19. Meselson & Weigle - part 2 Note the recombined viral genomes The process was enhanced by UV light treatment (causes DNA nicking) Recovery of intermediate density phage is proof that recombination occurred

20. Note the recombined viral genomes The process was enhanced by UV light treatment (causes DNA nicking) Recovery of intermediate density phage is proof that recombination occurred Lawn of host cells Plaque assay (1 phage infects one cell) Heavy XYZ Light xyz Intermediate density phage Examine virus from single plaque…. … progeny viruses sometimes had 2 different genomes! Start with two different phage genotypes: XYZ and xyz Mechanistic clue: it’s not always just cutting and pasting (got “heteroduplex” recombinant genomes)

21. How you can explain the results Note the recombined viral genomes The process was enhanced by UV light treatment (causes DNA nicking) Recovery of intermediate density phage is proof that recombination occurred

22. A single recombination experiment can give two different types of recombination products The process was enhanced by UV light treatment (causes DNA nicking) Recovery of intermediate density phage is proof that recombination occurred Splice recombinant Patch recombinant Two different Starting genotypes

23. Mechanism of Recombination General recombination: any pair of homologous DNA segments as substrates (100% homology NOT needed) In 1964, Robin Holliday proposed a model involving single-stranded nicks at homologous sites Duplex unwinding, strand invasion and ligation create a Holliday junction

24. Recombination Model 1) Alignment of 2 homologous DNA duplexes 2) single-stranded nick occurs 3) Strand exchange/ invasion 4) exchanged strands are ligated together & form a Holliday junction 5) junction migrates causing recombination of the two duplex DNAs

25. Recombination Model (continued) 6) Resolution of the junction intermediate gives either patch or splice recombinants

26. Resolution of Holliday Junctions “Patch” “Splice”

27. Resolution of Holliday Junctions To see this in 3D go to: http://www.wisc.edu/genetics/Holliday/holliday3D.html

28. Requirements for Recombination 1) Initiate the process -recombination requires a single-stranded DNA overhang gap Either: -2 enzymatic activities: 1) helicase: unwinds duplex DNA, ATP-dependent 2) nuclease: DNA hydrolysis (breaking backbone) -overhangs are produced by RecBCD Enzyme complex = RecB, RecC and RecD proteins

29. Chi sequence: 5’-GCTGGTGG-3’ ~1000 such sites on the E. coli genome -are recombinational hot-spots Single-Stranded Binding protein (SSB) binds ssDNA non-specifically and protects it from degradation, and from becoming double stranded again 1) Initiate

30. Requirements for Recombination 2) Holliday junction formation -by RecA - has “recombinase” activity - Mediates homologous base pairing (aligns 2 homologous DNA partners) - Catalyzes strand exchange, ATP-dependent

31. 2) Strand exchange and junction formation

32. The RecA Protein 38 kD enzyme that catalyzes ATP- dependent DNA strand exchange, leading to formation of Holliday junction RecA forms a helical filament with a groove to accommodate DNA

33. RecA protein Filament crystal structure A single RecA = 38kD (ribbon diagram and red monomer) Can assemble into a Helical Filament = 6 RecA’s per turn Helical filament has a groove that can fit DNA DNA modeled into the groove has to have its normal helix extended to 150% normal length

34. RecA has 2 sites for binding to DNA: 1 o site and 2 o site The 1 o site has a higher affinity for DNA, so it gets filled first. Next, the 2 o site is filled by the recombination partner dsDNA. But this binding is transient i.e. the RecA is scanning along the dsDNA. If the single strand in the 1 o site can form a duplex with one strand of the recombination partner then the remaining single strand gets trapped tightly in site 2. Why? A. site 2 has a high affinity for ssDNA

35. Model for DNA Structure During Strand Exchange Proc. Natl. Acad. Sci. USA Vol. 98, Issue 15, 8425-8432, July 17, 2001 “Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51 family of proteins: a possible advantage of DNA over RNA as genetic material” Takehiko Shibata et al.

36. used NMR to investigate structure of RecA- ssDNA complexes in solution Mix: RecA protein ssDNA (short oligonucleotide) ATP  S (non-hydrolyzable analog of ATP) Take NMR spectrum Concluded: when bound to RecA the ssDNA forms a helix that is 1.5X length of B form DNA

37. when bound to RecA the ssDNA forms a helix that is 1.5X length of B form DNA modeled that would be same for the dsDNA adjacent bases are too far apart to stack so what stabilizes the helix??? Significance of the conclusions van der Waals interactions between the sugar 2’C (methylene group) of one nucleotide and the adjacent base stabilize the helix. This gives rotational flexibility to the bases in the DNA

38. Significance of the conclusions van der Waals interactions between the sugar 2’C (methylene group) of one nucleotide and the adjacent base stabilize the helix. This gives rotational flexibility to the bases in the DNA

39. RecA-bound DNANormal B form DNA 2’ methylene group RNA can’t do this because of the bulky hydroxyl group at the 2’ C

40. Question: What drives the base rotation?? Answer: Conversion of sugar puckers

41. Finishing Off Recombination RecA starts branch migration RuvA, RuvB, drive branch migration and RuvC processes the Holliday junction into recombination products

42. RuvA - is a specificity factor - it recognizes the junction and binds to it RuvB - is an ATP-dependant motor - it migrates the junction - Junction resolution is done by RuvC -an endonuclease

43. Efficient Branch Migration Accomplished by a complex of RuvA/RuvB RuvA (crystal structure was solved in 1996) functions as a tetramer binds to Holliday junction structure has a core of negatively charged amino acids that force apart the DNA strands at the junction center facilitates binding of RuvB

44. The RuvA tetramer Ribbon structureCharge distributionSpace-fill model + DNA

45. Efficient Branch Migration Accomplished by a complex of RuvA/RuvB RuvB = ATP-dependent helicase forms a ring of 6 monomers surrounds the heteroduplex DNA one RuvB ring assembles on either side of the Holliday junction drives migration by pulling ds DNA through the rings over the RuvA core

46. Migration of Holliday Junctions To see this in motion go to: http://www.sdsc.edu/journals/mbb/ruva.html

47. The RuvC endonuclease

48. So much for bacteria, What about us?? proteins with recA activity exist in eukaryotes (from yeast to mammals) examples are: Rad51, Rad55, Rad57, DncI Function in DNA repair Can mediate homologous strand exchange in vitro Form nucleoprotein filaments just like RecA

49. For next class We will switch chapters We are done with recombination (no transposons, no Immunoglobulin genes) Please read: Chapter 30 sections 30.1, 30.2, 30.3