Abstract:

A challenge for chromosome segregation in all domains of life is the formation of catenated progeny chromosomes, which arise during replication as a consequence of the interwound strands of the DNA double helix. Topoisomerases play a key role in DNA unlinking both during and at the completion of replication. Here we report that chromosome unlinking can instead be accomplished by multiple rounds of site-specific recombination. We show that step-wise, site-specific recombination by XerCD-dif or Cre-loxP can unlink bacterial chromosomes in vivo, in reactions that require KOPS-guided DNA translocation by FtsK. Furthermore, we show that overexpression of a cytoplasmic FtsK derivative is sufficient to allow chromosome unlinking by XerCD-dif recombination when either subunit of TopoIV is inactivated. We conclude that FtsK acts in vivo to simplify chromosomal topology as Xer recombination interconverts monomeric and dimeric chromosomes.

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Unlinking chromosome catenanes <em>in vivo</em> by site-specific recombination

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^{Department of Biochemistry, University of Oxford, Oxford, UK}

^{Department of Mathematics, San Francisco State University, San Francisco, CA, USA}

^{Division of Molecular Genetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Tel.: +44 1865 275296; Fax: +44 1865 275297; E-mail:}ku.ca.xo.hcoib@ttarrehs

^{These authors contributed equally to this work}

^{Present address: Cancer Research UK, Clare Hall Laboratories, London Research Institute, Herts EN6 3LD, UK}

Received 2007 Jul 13; Accepted 2007 Aug 14.

Abstract

A challenge for chromosome segregation in all domains of life is the formation of catenated progeny chromosomes, which arise during replication as a consequence of the interwound strands of the DNA double helix. Topoisomerases play a key role in DNA unlinking both during and at the completion of replication. Here we report that chromosome unlinking can instead be accomplished by multiple rounds of site-specific recombination. We show that step-wise, site-specific recombination by XerCD-dif or Cre-loxP can unlink bacterial chromosomes in vivo, in reactions that require KOPS-guided DNA translocation by FtsK. Furthermore, we show that overexpression of a cytoplasmic FtsK derivative is sufficient to allow chromosome unlinking by XerCD-dif recombination when either subunit of TopoIV is inactivated. We conclude that FtsK acts in vivo to simplify chromosomal topology as Xer recombination interconverts monomeric and dimeric chromosomes.

Keywords: chromosome segregation, decatenation, FtsK, tangles, XerCD

Abstract

Click here to view.^{(43K, doc)}

Acknowledgments

We thank K Marians for supplying the pLex-dnaX(γ) plasmid, O Espeli for assistance with the parEts rescue protocol, Sean Colloms and Marshall Stark for the Cre expression vectors pMS173 and pMS174, and L Zechiederich for the C600parC1215 and W3110parE10 strains. We thank S Trigueros for valuable discussion. The research was supported by the Wellcome Trust. VS was supported by a Clarendon scholarship. MV was supported by Grant P20 MD000262 from RIMI (NCMHD, NIH) and by NIH SCORE 506 {"type":"entrez-nucleotide","attrs":{"text":"GM052588","term_id":"218074608","term_text":"GM052588"}}GM052588.

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