Plasmid RK2 ParB protein: purification and nuclease properties.
The parCBA operon of the 3.2-kb stabilization region of plasmid RK2 encodes three cotranslated proteins. ParA mediates site-specific recombination to resolve plasmid multimers, ParB has been shown to be a nuclease, and the function of ParC is unknown. In this study ParB was overexpressed by cotranslation with ParC in Escherichia coli by using a plasmid construct that contained the parC and parB genes under the control of the T7 promoter. Purification was achieved by treatment of extracts with Polymin P, followed by ammonium sulfate precipitation and heparin and ion-exchange chromatography. Sizing-column analysis indicated that ParB exists as a monomer in solution. Analysis of the enzymatic properties of purified ParB indicated that the protein preferentially cleaves single-stranded DNA. ParB also nicks supercoiled plasmid DNA preferably at sites with potential single-stranded character, like AT-rich regions and sequences that can form cruciform structures. ParB also exhibits 5'-->3' exonuclease activity. This ParB activity on a 5'-end-labeled, double-stranded DNA substrate produces a 3', 5'-phosphorylated dinucleotide which is further cleaved to a 3', 5'-phosphorylated mononucleotide. The role of the ParB endonuclease and exonuclease activities in plasmid RK2 stabilization remains to be determined.
Johnson
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Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0322, USA.
Mincer
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Schwab
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Burgin
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Helinski
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D001483:Base Sequence; D019915:DNA Primase; D027101:DNA Topoisomerase IV; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004720:Endonucleases; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005092:Exonucleases; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D019915:DNA Primase; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C121791:dnaG protein, E coli; D043211:Exodeoxyribonuclease V; D027101:DNA Topoisomerase IV; D004250:DNA Topoisomerases, Type II