1887

Abstract

The operon, which together with the operon constitutes an efficient stabilization system of the broad-host-range plasmid RP4, encodes a 20 kDa polypeptide (ParB), which exhibits sequence homology to nucleases. The ParB protein was overexpressed by means of an inducible -promoter system. Plate assays with herring sperm DNA as substrate provided evidence for nuclease activity. The ParB nuclease shows specificity for circular DNA substrates and linearizes them regardless of the presence of parts of the RP4 partitioning region. The nuclease activity is stimulated by the presence of Ca ions. EDTA (5 mM) completely inhibits nuclease activity. By restriction analysis of the ParB-linearized products, cleavage of circular DNA substrates taking place preferentially at specific sites was demonstrated. Run-off sequencing and primer extension analysis of ParB-linearized plasmid DNA revealed a specific target for ParB action adjacent to an AT-rich region containing palindromic sequence elements on a pBR322-derived plasmid.

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1997-12-01
2021-07-29
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