The ParB protein encoded by the RP4 par region is a Ca(2+)-dependent nuclease linearizing circular DNA substrates.

Microbiology
Elisabeth GrohmannHelmut Schwab

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.

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Citations

Mar 2, 2016·Proceedings of the National Academy of Sciences of the United States of America·Jennifer J ThiavilleValérie de Crécy-Lagard
Mar 28, 2012·Protein Expression and Purification·Kunyuan GuoXinxin Zhang
Jun 21, 2016·Chemical Reviews·Peter Weigele, Elisabeth A Raleigh
Sep 28, 1999·Journal of Bacteriology·E P JohnsonD R Helinski
Nov 13, 1998·Journal of Bacteriology·C L EasterD R Helinski

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