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J Biol Chem, Vol. 275, Issue 14, 9930-9936, April 7, 2000
From the Division of Molecular Genetics, Department of
Biochemistry, University of Oxford, South Parks Road, Oxford OX1
3QU, United Kingdom
Successful segregation of circular chromosomes in
Escherichia coli requires that dimeric replicons, produced
by homologous recombination, are converted to monomers prior to cell
division. The Xer site-specific recombination system uses two related
tyrosine recombinases, XerC and XerD, to catalyze resolution of
circular dimers at the chromosomal site, dif. A 33-base
pair DNA fragment containing the 28-base pair minimal dif
site is sufficient for the recombinases to mediate both inter- and
intramolecular site-specific recombination in vivo. We show
that Xer-mediated intermolecular recombination in vitro
between nicked linear dif "suicide" substrates and
supercoiled plasmid DNA containing dif is initiated by
XerC. Furthermore, on the appropriate substrate, the nicked Holliday junction intermediate formed by XerC is converted to a linear product
by a subsequent single XerD-mediated strand exchange. We also
demonstrate that a XerC homologue from Pseudomonas
aeruginosa stimulates strand cleavage by XerD on a nicked linear
substrate and promotes initiation of strand exchange by XerD in an
intermolecular reaction between linear and supercoiled DNA, thereby
reversing the normal order of strand exchanges.
Sequential Strand Exchange by XerC and XerD during Site-specific
Recombination at dif*
,, and
*
This work was supported by Wellcome Trust Program Grant
052152/Z/97 (to D. J. S.) and Career Development Fellowship
039542/A/98 (to G. W. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Inst. of Cell and Molecular Biology, University
of Edinburgh, Kings Bldg., Edinburgh EH9 3JR, UK.
§
To whom correspondence should be addressed. Tel.: 44-01865-275296;
E-mail: sherratt@bioch.ox.ac.uk.
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