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The ParB protein encoded by the RP4 par region is a Ca²⁺-dependent nuclease linearizing circular DNA substrates

Elisabeth Grohmann

Institut für Biotechnologie, Arbeitsgruppe Genetik, Technische Universität Graz, Petersgasse 12, A-8010 Graz, Austria

Author for correspondence: Helmut Schwab. Tel: +43 316 873 8418. Fax: +43 316 873 8434. e-mail: SCHWAB@BIOTE.TU-GRAZ.AC.AT

ABSTRACT

The parCBA operon, which together with the parDE 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 tac-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 in cis of parts of the RP4 partitioning region. The nuclease activity in vitro is stimulated by the presence of Ca²⁺ 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.

Present address: Centro de Investigaciones Biologicas, C/Velazquez, 144, 28006, Madrid, Spain.

This article has been cited by other articles:

				E. P. Johnson, T. Mincer, H. Schwab, A. B. Burgin, and D. R. Helinski Plasmid RK2 ParB Protein: Purification and Nuclease Properties J. Bacteriol., October 1, 1999; 181(19): 6010 - 6018. [Abstract] [Full Text]

				C. L. Easter, H. Schwab, and D. R. Helinski Role of the parCBA Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance J. Bacteriol., November 15, 1998; 180(22): 6023 - 6030. [Abstract] [Full Text]