Plasmid RK2 ParB protein: purification and nuclease properties

Abstract

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.

FIG. 1

Purification of the ParB protein. Fractions of material from the purification steps described in Materials and Methods were analyzed by SDS-PAGE. The gels were stained with Coomassie brilliant blue R. The positions of ParB and ParC are indicated. Lane 1, molecular weight standards (LMW); lanes 2 and 3, total SDS-soluble protein of uninduced and induced cultures, respectively; lane 4, supernatant after sonication; lane 5, supernatant after treatment with Polymin P; lane 6, material after dialysis of the Polymin P supernatant; lanes 7 and 8, peak fractions after chromatography on heparin and Mono-S columns, respectively.

FIG. 2

Superose-12 gel filtration of purified ParB. ParB, ovalbumin (44 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.5 kDa) were mixed and subjected to Superose-12 gel filtration. The column fractions are indicated at the bottom, and the position of each protein is indicated on the right.

FIG. 3

Time course treatment of pUC19 DNA by ParB. Supercoiled pUC19 DNA was treated with ParB for 0, 0.5, 1, 2, 4, or 8 min (lanes 2 to 6, respectively) as described in Materials and Methods. The positions of the various forms of pUC19 are indicated.

FIG. 4

Restriction enzyme cleavage of ParB-linearized pUC19 DNA. Supercoiled DNA was untreated (lanes 1 to 4) or treated with ParB (lanes 5 to 8) or with EcoRI (lanes 9 to 11). Samples were then analyzed without an additional digestion (lanes 1 and 5) or after digestion with SspI (lanes 2, 6, and 10), DraI (lanes 3, 7, and 11), or EcoRI (lanes 4 and 8). Agarose gel electrophoresis was carried out as described in Materials and Methods.

FIG. 5

Primer extension of ParB-treated supercoiled DNA. (A) pUC19 DNA was treated with FspI and AlwNI (lanes 1 and 4) or with ParB to produce open circular (oc) (lanes 2 and 5) or linear (lanes 3 and 6) DNA. Primer extension was then performed with radiolabeled primers against the top strand (lanes 1 to 3) and against the bottom strand (lanes 4 to 6) of the treated pUC19. The hatched boxes indicate the position of the AT-rich region. (B) Results of primer extension performed on the top strand of the FspI-AlwNI fragment of pUC19 and on ParB-treated open circular and linear DNAs with primer 1293TSpUC (right panel) and results of sequencing with the same primer (left panel). (C) Results of primer extension performed on the bottom strand of the FspI-AlwNI fragment of pUC19 and on ParB-treated open circular and linear DNAs with primer 1484BSpUC (right panel) and sequencing of this region with the same primer (left panel).

FIG. 6

Preferred ParB-nicked sites in pUC19 DNA. (A) Sites of preferred ParB nicking adjacent to the DraI restriction enzyme sites. By using Oligo 5.0, the internal binding stabilities of 10-bp windows of base pairs within the pUC19 sequence were calculated. The nucleotide sequence of the DraI region is shown at the bottom. A point along the x axis of the graph corresponds to the stability of a 10-bp window beginning with the nucleotide shown directly below it. The position on the graph indicated with an open arrowhead corresponds to the 10-bp sequence underlined. The filled arrowheads indicate sites of preferred cleavage by ParB suggested by analysis of the top strand. The dashed line indicates the average stability of 10-bp windows of the entire pUC19 sequence. The positions of the DraI restriction sites are also indicated. (B) Site of preferred ParB nicking within the bla gene of pUC19. The sequences making up the cruciform structure and the positions on the pUC19 map are shown. The open arrowhead indicates the position of preferred ParB cleavage on the top strand. The filled arrowhead indicates the position of preferred ParB cleavage on the bottom strand.

FIG. 7

ParB activity on double-stranded and single-stranded DNAs. Double-stranded and single-stranded pBluescript II SK(−) DNAs were treated with ParB for 5 min. Double-stranded DNA was treated with 0, 250, 500, 1,000, 5,000, or 10,000 pg of ParB (lanes 1 to 6), while single-stranded DNA was treated with 0, 2.5, 5, 10, 50, and 100 pg of ParB (lanes 7 to 12). The positions of the open circular (oc), linear (l), and supercoiled (ccc) DNA forms are shown.

FIG. 8

Exonuclease assay. A 20-bp double-stranded DNA fragment was 5′ radiolabeled on one end and then treated with 0.01, 0.1, or 1 U of exonuclease III (lanes 2 to 4, respectively); with 0.1, 1, or 2 U of the T7 gene 6 exonuclease (lanes 5 to 7, respectively); or with 0.1, 1, 10, 20, or 40 ng of purified ParB (lanes 8 to 12, respectively). The positions of AMP, ADP, and ATP are indicated.