ABSTRACT
Early steps of retroviral replication involve reverse transcription of the viral RNA genome and integration of the resulting cDNA copy into a chromosome of the host cell. The initial DNA breaking and joining steps of integration are carried out by the virus-encoded integrase enzyme. Integrases bind specifically to the ends of the unintegrated viral cDNA but nonspecifically to target DNA. Conventional assays in vitro reveal primarily the nonspecific DNA binding mode, complicating studies of integrase--DNA complexes. Here, we report an investigation of unconventional DNA structures useful for positioning integrase at predetermined sites. We find that paired DNA three-way junctions can be used to mimic branched DNAs normally formed as reaction intermediates. The three-way junctions differ from authentic intermediates in the connectivity of the DNAs, which, in contrast to the authentic intermediate, allow formation of stable DNA structures under physiological conditions. Assays in vitro showed that integrase can direct hydrolysis at sequences resembling the viral cDNA ends within the three-way junction, but not on junctions with mutant sequences. Changing the spacing between the paired three-way junctions disrupted the cleavage pattern, emphasizing the importance of the correct DNA scaffold. DNase I footprinting studies revealed protection of specific bases at the terminus of the LTR in the three-way junction complex, but not on control linear DNA, specifying the locations of tight interactions between integrase and DNA. Paired DNA three-way junctions are attractive reagents for structural studies of integrase-DNA complexes.
Subject(s)
Avian Sarcoma Viruses/genetics , DNA, Viral/chemistry , DNA, Viral/metabolism , HIV-1/genetics , Integrases/metabolism , Avian Sarcoma Viruses/metabolism , Base Sequence , DNA Footprinting , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Complementary/metabolism , DNA, Viral/genetics , HIV Integrase/metabolism , HIV-1/metabolism , Hydrolysis , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Terminal Repeat Sequences/genetics , Terminal Repeat Sequences/physiologyABSTRACT
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.
Subject(s)
Endodeoxyribonucleases/isolation & purification , Escherichia coli Proteins , Exodeoxyribonucleases/isolation & purification , Plasmids , Base Sequence , DNA Primase , DNA Topoisomerase IV , DNA Topoisomerases, Type II/biosynthesis , DNA Topoisomerases, Type II/genetics , DNA, Single-Stranded/metabolism , DNA, Superhelical/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Endonucleases , Escherichia coli/genetics , Exodeoxyribonuclease V , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Exonucleases , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
The parDE operon, located within the 3.2-kb stabilization region of plasmid RK2, encodes antitoxin (ParD) and toxin (ParE) proteins that stabilize the maintenance of this broad-host-range plasmid via a postsegregational killing mechanism. A ParE protein derivative, designated ParE', was purified by construction of a fusion protein, GST-ParE, followed by glutathione-agarose binding and cleavage of the fusion protein. ParE' has three additional amino acids on the N terminus and a methionine residue in place of the native leucine residue. The results of glutathione-agarose affinity binding and glutaraldehyde cross-linking indicate that ParE' exists as a dimer in solution and that it binds to the dimeric form of ParD to form a tetrameric complex. The formation of this complex is presumably responsible for the ability of ParD to neutralize ParE toxin activity. Previous studies demonstrated that the parDE operon is autoregulated as a result of the binding of the ParD protein to the parDE promoter. ParE' also binds to the parDE promoter but only in the presence of the autoregulatory ParD protein. ParE', in the presence or absence of the ParD protein, does not bind to any other part of the 3.2-kb stabilization region. The binding of the ParE' protein to ParD did not alter the DNase I footprint pattern obtained as a result of ParD binding to the parDE promoter. The role of ParE in binding along with ParD to the promoter, if any, remains unclear.
Subject(s)
Bacterial Proteins/genetics , Bacterial Toxins/genetics , DNA-Binding Proteins/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Plasmids/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Bacterial Toxins/isolation & purification , Bacterial Toxins/metabolism , DNA Topoisomerase IV , DNA, Bacterial/metabolism , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Glutathione Transferase/biosynthesis , Glutathione Transferase/genetics , Promoter Regions, Genetic , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Restriction Mapping , Transformation, GeneticABSTRACT
Over the past 3 years, 15 successful nurse managers have been chosen using an objective selection process developed by the authors. A process to help the nurse executive answer the question, Am I choosing the right person for this job? is described. Through the use of an applicant assessment process, a candidate's leadership ability is revealed, and subjectivity in selection is considerably reduced.
