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Genetic Recombination

Purposes Promotes genetic diversity within a species - within a chromosome causes inversions, deletions, duplications - horizontal exchange introduces new sequences (information). In the lab:. introduce foreign DNA or mutations into bacteria.

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Genetic Recombination

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  1. Purposes • Promotes genetic diversity within a species • - within a chromosome causes inversions, deletions, duplications • - horizontal exchange introduces new sequences (information) In the lab: introduce foreign DNA or mutations into bacteria map the distance between mutations Genetic Recombination Definition: The breakage and joining of DNA into new combinations • Plays a major role in repair of damaged DNA and mutagenesis • Critical for several mechanisms of phase and antigenic variation

  2. Types: • Homologous recombination (or general recombination) • basic steps • current models • proteins that play a role • practical applications • Nonhomologous recombination (site-specific) • Basic steps • general categories of proteins used • examples – phage integration, flagellin phase variation • Illegitimate recombination (transposition)

  3. CACATGATACGTCCGATCACATTTGTTGTTCATAT GTGTACTATGCAGGCTAGTGTAAACAACAAGTATA GTGTACTATGCAGGCTAGTGTAAACAACAAGTATA CACATGATACGTCCGATCACATTTGTTGTTCATAT • Results in the creation of a synapse [synapse is point where DNA strands are held • together by complementary base-pairing (H bonds)] CACATGATACGTCCGATCACATTTGTTGTTCATAT GTGTACTATGCAGGCTAGTGTAAA Synapse C GTATA A A A A C GTATA GTGTACTATGCAGGCTAGTGTAAA CACATGATACGTCCGATCACATTTGTTGTTCATAT Homologous Recombination • Step One • Formation of complementary base pairing between two ds DNA molecules - Sequences must be the same or very similar - 23 base pair minimum

  4. CACATGATACGTCCGATCACATTTGTTGTTCATAT GTGTACTATGCAGGCTAGTGTAAA C GTATA CACATGATACGTCCGATCACATTTGTTGTTCATAT GTGTA A A A A GGCTAGTGTAAACAACAAGTATA C G A C T GTATA GTGTACTATGCAGGCTAGTGTAAA A C CACATGATACGTCCGATCACATTTGTTGTTCATAT T G G T Branch migration A C T A C GGCTAGTGTAAACAACAAGTATA GTGTA CACATGATACGTCCGATCACATTTGTTGTTCATAT Step two Branch migration to extend the region of base-pairing (the heteroduplex) • ATP-hydrolyzing proteins (Ruv proteins) break and re-form H bonds • allow migration to go faster

  5. CACATGATACGTCCGATCACATTTGTTGTTCATAT GTGTACTATGCAGGCTAGTGTAAA C GTATA CACATGAT ACGT CCGATCACATTTGTTGTTCATAT GTGTA A A A A GGCTGGTGTAAACAACAAGTATA C G A C T GTATA GTGTACTATGCAGGCTGGTGTAAA A C CACATGATACGTCCGACCACATTTGTTGTTCATAT T G G T Branch migration A C T A C GGCTAGTGTAAACAACAAGTATA GTGTA CACATGAT AC GT CCGACCACATTTGTTGTTCATAT Branch extension can increase the chance of gene conversion via increasing the chances of including mismatches in the heteroduplex region

  6. Step three Resolution of the heteroduplex - isomerization of the duplex due to strands uncrossing and re-crossing - results in different outcomes upon resolution - 50% chance of each isomer being resolved

  7. Models of Homologous Recombination (I) Holliday double-strand invasion Fig. 10.1 of textbook • Initiated by two single-stranded breaks made simultaneously and exactly • in the same place in the DNA molecules to be recombined • Free ends of the two broken strands cross over each other, pairing with its • complementary sequence in the other DNA molecule to form heteroduplex. • See http://engels.genetics.wisc.edu/Holliday/holliday3D.html for strand resolution

  8. Models of Homologous Recombination (II) Single-strand Invasion • Single strand break in one molecule • Free ss end invades other DNA molecule • Gap on cut DNA is filled in by • DNA polymerase • Displaced strand on other DNA molecule • is degraded • Two ends are joined • Initially, heteroduplex is only on • one strand; branch migration causes • another heteroduplex on other molecule Fig. 10.3 of textbook

  9. Models of Homologous Recombination (III) Double strand break-repair • A double stranded break occurs in one • molecule; and exonuclease digests the • 5’ ends of each break, leaving a gap • One of the 3’ tails invades unbroken • molecule; pairs with complementary • sequence • DNA polymerase extends the tail until • it can be joined with 5’ end • Displaced strand used as template to • replace gap on other molecule • Two Holliday junctions form (may • produce recombinant flanking DNA • depending how they are resolved) Fig. 10.4 of textbook

  10. + or Donor DNA Mutant bank (i.e. of E. coli) Screen for inability to acquire a selectable marker Proteins involved in DNA recombination (the E. coli paradigm) Mutation Phenotype RecA RecBC RecD RecF RecJ RecO RecR RecQ RecN RecG RuvA RuvB RuvC PriA PriB PriC DnaT Recombination deficient Reduced recombination Rec+  independent Reduced plasmid recombination • Reduced recombination in RecBC- • as above • as above • as above Reduced recombination in RecBC- Reduced recombination in RuvA-B-C- Reduced recombination in RecG- as above as above Reduced recombination as above as above as above

  11. Chi (Χ) site: • 8 base pair sequence without • symmetry (5’GCTGGTCC) • Greatly stimulates ability of RecBCD • to catalyze recombination RecBCD exonuclease: opens strands • RecBCD binds to DNA at one end or at • a ds breakage point • Moves along the DNA, creating a loop • and degrading the strand with a free • 3’ end via its exonuclease activity • Exonuclease activity is inhibited • upon passing a Chi site of orientation • Upon cessation of exonuclease activity, • the undegraded 3’ end pairs with • homologous sequences on another • DNA molecule

  12. RecA: needed to form triple helix • RecA binds to free strand to form an • extended helical structure. • Resultant DNA-RecA helix forms a • triple-stranded helix with ds DNA • that has a homologous region • one of the strands in the ds helix is • displaced (D loop) • displaced strand binds to original • complementary strand of the invasive • strand to create Holliday junction

  13. RecA protein-dsDNA complex imaged by atomic force microscopy www-mic.ucdavis.edu

  14. RecF pathway • important for DNA repair • (i.e. UV light) • detectable as reduced • recombination in RecBC- • background • Important after heteroduplex • formation is initiated • branch migration • resolution of heterduplex Proteins involved in DNA recombination (the E. coli paradigm) (con’t) Mutation Phenotype RecA RecBC RecD RecF RecJ RecO RecR RecQ RecN RecG RuvA RuvB RuvC PriA PriB PriC DnaT Recombination deficient Reduced recombination Rec+  independent Reduced plasmid recombination • Reduced recombination in RecBC- • as above • as above • as above Reduced recombination in RecBC- Reduced recombination in RuvA-B-C- Reduced recombination in RecG- as above as above Reduced recombination as above as above as above

  15. RuvB RuvA RuvC resolves (cuts) the Holliday junction • Ruv C is a specialized endonuclease • an X-phile – cuts crossed DNA strands • always cuts at two T’s Efficient branch migration requires RuvA and RuvB • RuvA specifically binds Holliday junctions • - resultant structure better able to undergo branch migration and resolution • RuvB is a helicase • - forms a hexameric ring around the DNA strand • - DNA is pumped through the ring using ATP cleavage to drive the pump • - the synapse is thus forced to migrate

  16. RuvA RuvB RuvB A simple model of a RuvA/RuvB/DNA complex extrapolating from the above model and in agreement with the electron microscopy results of Parsons et al. (Nature 374, 375 (1995)). RuvA binds the Holliday junction at the central crossover point and targets two RuvB hexamers onto opposite arms of the DNA where they encircle the DNA duplexes and facilitate branch migration in concert with RuvA in an ATP dependent manner. For animation, see http://www.sdsc.edu/journals/mbb/ruva.html

  17. AmpR EmR b’ a a’ b Or b’ EmR a a a a 5’ end of gene a a Single cross-over results in one truncated copy and one intact copy of the gene a a internal fragment Single cross-over results in an interrupted gene a b a’ AmpR

  18. a a a a Single cross-over outcome when using one end of the gene a a b P2 P1 a a b b P1 a a P2 Useful for introducing a promoter-reporter gene fusion without disrupting the gene’s function. b

  19. Example: phage integrases Example: Salmonella flagellin phase variation Nonhomologous (Site-specific) Recombination • Occurs at specific or highly preferred target and donor DNA sequences • Requires special proteins that recognize specific sequences and • catalyze the molecular events required for strand exchange • Relatively rare compared to homologous recombination Site-specific recombinases include: - integrases recognize and promote recombination between two sequences of DNA - resolvases resolve co-integrates by pairing sequences within sites that are present in direct orientation to each other (example - transposon resolvases) - invertases pair sequences within sites that are present in reverse orientation to each other intermolecular intramolecular

  20. Lytic/Lysogenic Developmental Switch

  21. Examples of site-specific recombination 1) Phage integration and excision • Integration of circular phage • DNAinto the host DNA to • form a prophage occurs via • the action ofphage Int • enzymes (integrases). • Usually highly specific and • occurs at only one or a few • integration sites on the • chromosome • Excision utilizes both the • integrase and an excisase, • which act at the hybrid • integration sites that flank the • prophage

  22. integrase excisase

  23. GCTTTTTTATACTAA Phage integration and excision (con’t) • Lysogenization by lambda phage: • Site-specific recombination between the attP site on phage and • the attB site on bacterial chromosome • attP and attB are dissimilar except for 15 bp core sequence • GCTTTTTTATACTAA • The lambda Int protein is an integrase that promotes site-specific • recombination between 7 internal bases of the core sequence • Excision is via production on integrase (Int) and excisase (Xis), which promote • recombination of the hybrid attP/B and attB/P molecules in the chromosome

  24. Lysogenic state

  25. hin H2 flagellin Repressor H1 flagellin Examples of site-specific recombination (con’t) 2) Phase variation of Salmonella flagellin genes • Reversible, high frequency (10-4) inversion of DNA sequence that carries • the promoter for one flagellin structural gene and for a repressor of • a second flagellin gene • Occurs by virtue of a DNA invertase called Hin • Promotes site-specific recombination between two closely linked • sites of DNA P Inverted repeats

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