title: The ParB protein encoded by the RP4 par region is a Ca(2+)-dependent nuclease linearizing circular DNA substrates.
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.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Grohmann
authors.affiliation: Institut f�r Biotechnologie, Arbeitsgruppe Genetik, Technische Universit�t Graz, Petersgasse 12, A-8010 Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Stanzer
authors.affiliation: Institut f�r Biotechnologie, Arbeitsgruppe Genetik, Technische Universit�t Graz, Petersgasse 12, A-8010 Graz, Austria.
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D003001:Cloning, Molecular; D004270:DNA, Circular / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005399:Fishes; D008297:Male; D008969:Molecular Sequence Data; D009876:Operon; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013094:Spermatozoa; D013210:Staphylococcus / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D004270:DNA, Circular; D011994:Recombinant Proteins; D003851:Deoxyribonucleases; D002118:Calcium