1887

Abstract

The plasmid pJHCMW1 encodes resistance to several aminoglycosides and β-lactams and consists of a copy of the transposon Tn, a region including the replication functions, and a sequence with homology to ColE1 , designated . In this work, the role of this -like site in ensuring the stable inheritance of pJHCMW1 by multimer resolution was studied. The Xer site-specific recombination system acts at sites such as ColE1 to resolve plasmid multimers formed by homologous recombination, thereby maintaining plasmids in a monomeric state and helping to ensure stable plasmid inheritance. Despite its high similarity to ColE1 , the pJHCMW1 was a poor substrate for Xer recombination in and did not contribute significantly to plasmid stability. Instead, the Tn co-integrate resolution system was highly active at resolving pJHCMW1 multimers and ensured the stable inheritance of pJHCMW1. Although Xer recombination at pJHCMW1 was inefficient in , the recombination that did occur was dependent on ArgR, PepA, XerC and XerD. A supercoiled circular DNA molecule containing two pJHCMW1 sites in direct repeat yielded Holliday-junction-containing product when incubated with ArgR, PepA, XerC and XerD , confirming that pJHCMW1 is a functional recombination site. However, unlike , some Holliday-junction-containing product could be detected for in the absence of ArgR, although addition of this protein resulted in formation of more Holliday junctions. Binding experiments demonstrated that XerD bound to pJHCMW1 core with a high affinity, but that XerC bound to this site very poorly, even in the presence of XerD.

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2000-03-01
2019-11-21
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