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FtsK-Dependent Dimer Resolution on Multiple Chromosomes in the Pathogen Vibrio cholerae

FtsK-Dependent Dimer Resolution on Multiple Chromosomes in the Pathogen Vibrio cholerae. PLoS Genetics 4 , 1-11 (2008). Molecular machines working on the chromsome. To ensure genetic materials passing on. Chromosome dimer resolution (CDR). FtsK. MM 64 , 1434–1441 (2007).

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FtsK-Dependent Dimer Resolution on Multiple Chromosomes in the Pathogen Vibrio cholerae

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  1. FtsK-Dependent Dimer Resolution on MultipleChromosomes in the Pathogen Vibrio cholerae PLoS Genetics4, 1-11 (2008)

  2. Molecular machines working on the chromsome To ensure genetic materials passing on

  3. Chromosome dimer resolution (CDR)

  4. FtsK MM64, 1434–1441 (2007)

  5. Segregation of the ter region during chromosome dimer resolution. MM64(6), 1434–1441 (2007)

  6. Model for SpoIIIE polarity MM66, 1066–1079 (2007)

  7. chrII, 1.07Mb Some essential genes and 13 TA loci Derived from megaplasmid? oriCIIvc: unique parAB2 cluster with plasmid Par chrI, 2.96Mb Most of the essential genes oriCIvc: oriC like parAB1 cluster to Chr encoded MM56, 1129–1138 (2005) Plasmid or schism hypothesis Chromosome initiate replication synchronously Nature406, 477-484 (2000)

  8. fdimerChrI+II Growth Competition (V. cholerae) f: frequency of cells that the mutant strains fail to produce at each generation compared to their parent Rate of dimer formation on ChrI/II, fdimerChrI/II Dimerformation on the two chromosomes is independent

  9. Visualizing the strand cleavage on suicide substrates • Generate DSB that prevents re-ligation • Accumulation of protein-5’-DNA complex • Accumulation of free 3’-DNA fragments

  10. Visualizing the strand cleavage on suicide substrates XerCVc cleaves the top strand XerDVc cleaves the bottom strands

  11. Positioning the cleavage sites XerCVc and XerDVc cleave DNA at the junction between their respective binding site and the central region of dif1 and dif2.

  12. product substrate + product % = FtsK, a Xer recombination activator In E.coli FtsKNEc background Para-FtsKVc dif1 or dif2 reporter plasmid 2hr 0.5% arabinose induction NdeI treatment substrate product Xer recombination at dif1 and dif2 depends on FtsKVc

  13. The order of strand exchanges? XerDVc mediate the first pair of strand exchanges HJ intermediates are preferentially resolved by XerCVc

  14. FtsKVc activates recombination at dif1 and dif2 by promoting the exchange of a first pair of strands by XerDVc

  15. Introduction on “Species specificity in Xer recombination activation”

  16. Species specificity in Xer recombination activation Species specificity is more pronounced on dif2 than on dif1.

  17. XerC binding central XerD binding ------------------ ---------- ------------------ ☆ ☆ Tight control of Xer recombination depends on the sequence of the resolution sites

  18. FtsK hybrids with N-terminal E.coli FtsK C-terminus Full length C-terminus C-terminal domain of FtsKVc completely rescue CDR in E. coli

  19. KOPS recognition FtsKVc directly recognizes the GGGCAGGG motif that engages N926, R930 and E933

  20. Candidate KOPS in V. cholerae GGGNAGGG motif might serve as KOPS in V. Cholerae

  21. Summary/ Discussion • Same FtsK dependent mechanism controls dimer resolution on each of the two V. cholerae chromosomes. • The particular genomic organization of the Vibrios seems to minimize chances for chromosome dimer formation. • A single pair of recombinase ensures dimer resolution of each of their non-homologous chromsomes. • Adoption of an FtsK-dependent dimer resolution system could be a key evolutionary step in the maintenance of large circular replicon • A few alterations in the sequence of dif1 and dif2 decreased the stringency of the control exerted by FtsKVc • Some selective pressure imposes the divergence of the central regions of CDR sites

  22. Progress Report

  23. merge YFP DAPI DIC ParA mutant localization correspond to nucleoid R31E K39E P-loop K44E ATP binding ATP hydrolysis G40V R247E D154A R218E D68A

  24. Some mutants affect ParA nucleoid targeting abilitybut some don’t. Do the mutants affect ParA-ParA or ParA-ParB interaction?  Bacterial two hybrid: evaluate protein-protein interaction in vivo

  25. Previous experiments showed a positive result only in the combination parA::pT18-pT25::parB parANC::pT18-pT25::parA Interacting with wild-type ParA R247E R218E D68A G40V

  26. interaction between mutants R247E R218E D68A All the mutants in the P-loop or ATP binding pocket lose their dimerization ability? Interaction between the two D68A mutants decreased significantly. Do ParA polymerization require hydrolysis ability?

  27. Interaction between ParA-ParB D68A R247E A-B R218E D68A, presumably an ATP hydrolysis mutant enhanced the miller reading. Indicating that ParA bind to ParB in its ATP bound form.

  28. Future plans Repeat the B2H experiment Plasmid stability assay Test the spore formation efficiency in S. coelicolor with ParA mutants The dynamic movement and filament forming ability of the mutants

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