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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) Speaker: 巫冠毅 R96B46021. Chromosome dimer resolution (CDR) -- Escherichia coli --. Inactivation of CDR caused 15% cell death per generation. ::. XerD. ::. KOPS.

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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) Speaker: 巫冠毅R96B46021

  2. Chromosome dimer resolution (CDR) --Escherichia coli -- Inactivation of CDR caused 15% cell death per generation

  3. :: XerD :: KOPS Architecture and domain structure of FtsK Ftsαβ form a hexamer that assembles around dsDNA β subdomain contains walker P-loop/ B motifs (1.6bp DNA displacement/ATP/monomer) γDomain confer DNA sequence specificity (toward dif) MM64, 1434–1441 (2007)

  4. Complex model of FtsKαβγ-KOPS MM64, 1434–1441 (2007)

  5. Model for DNA translocation directionality DNA is translocated opposite to the permissive direction of FtsK/SpoIIIE Nat. Struct. Mol. Biol. 15, 485-493(2008)

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

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

  8. Genome of Vibrio cholerae chrII, 1.07Mb (GC%=47.7) Some essential genes and 13 TA loci Derived from megaplasmid? oriCIIvc: unique (GC skew predicted) parAB2 cluster with plasmid Par chrI, 2.96Mb (GC%=46.9) Most of the essential genes oriCIvc: oriCEclike parAB1 cluster to Chr encoded 1. Plasmid or schism hypothesis 2. Two different replication initiation mechanism are synchronized 3. Once per cell cycle replication MM56, 1129–1138 (2005) Nature406, 477-484 (2000)

  9. FtsK dependent/independent Xer recombination • The sequence divergence in the central region of ChrII suggest a plasmid pathway • Position of XerDVc cleavage site on dif1 might differs from the dif Ec

  10. 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 Dimer formation on the two chromosomes is independent

  11. 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

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

  13. 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.

  14. 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 Plasmid extraction and NdeI treatment substrate product Xer recombination at dif1 and dif2 depends on FtsKVc

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

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

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

  18. XerC binding central XerD binding ------------------ ---------- ------------------ ☆ ☆ FtsK independent recombination

  19. Growth competition of FtsK hybrids with N-terminal E.coli FtsK C-terminus Full length C-terminus f%: frequency of cells that the parental N strain fails to produce at each generation compared to the FtsK hybrids C-terminal domain of FtsKVc completely rescue CDR in E. coli

  20. KOPS recognition Conservation in the KOPS recognition residues implies targeting to similar motifs FtsKVc directly recognizes the GGGCAGGG motif that engages N926, R930 and E933

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

  22. 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

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