Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference |
Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21 DE3 AI | Invitrogen | C607003 | Protein expression strain |
| Recombinant DNA reagent | pET11a | EMD Millipore | 9436 | Protein expression vector |
| Recombinant DNA reagent | pET28a-parSF | This work | Tether DNA PCR template | |
| Recombinant DNA reagent | pET28a | EMD Millipore | 69865 | Tether DNA PCR template |
| Recombinant DNA reagent | pBlueScript II KS(+) | Agilent | 212207 | Tether DNA PCR template |
| Recombinant DNA reagent | pX7 | Vecchiarelli et al., 2013 | ParAFoverexpression plasmid | |
| Recombinant DNA reagent | pX2 | Vecchiarelli et al., 2013 | ParAF-eGFP overexpression plasmid | |
| Recombinant DNA reagent | pX8 | Vecchiarelli et al., 2013 | ParBFoverexpression plasmid | |
| Recombinant DNA reagent | pET11a-ParBFR121A | This work | ParBFR121overexpression plasmid | |
| Recombinant DNA reagent | pET11a-ParBF1-42-mCherry | This work | ParBF1-42-mCherry overexpression plasmid | |
| Recombinant DNA reagent | pET11a-ParBF1-42 R36A-mCherry | This work | ParBF1-42 R36A-mCherry overexpression plasmid | |
| Recombinant DNA reagent | pET11a-ParBF1-42-mCherry-EcoRIE111Q | This work | ParBF1-42-mCherry-EcoRIE111Qoverexpression plasmid | |
| Sequence-based reagent | parSF DNA | This work | 5'-AGTCTGGGACCACGGTCCCACTCG | |
| Sequence-based reagent | parSF DNA Alexa 488 | This work | 5'-Alexa488-(HNS)-AGTCTGGGACCACGGTCCCACTCG | |
| Sequence-based reagent | parSF DNA complement strand | This work | 5'-CGAGTGGGACCGTGGTCCCAGACT | |
| Sequence-based reagent | Scrambled seq DNA | This work | 5'-AGTCTGCAGCTACTATACCACTCG | |
| Sequence-based reagent | Scrambled seq DNA complement strand | This work | 5'-CGAGTGGTATAGTAGCTGCAGACT | |
| Sequence-based reagent | EcoR1 sequence + strand | This work | 5'-GAATTCCGAGTGGGACCGTGGTCCCAGTCTGATTATCAGACCGAGAATTCAAGTTGGGACCGTGGTCCCAAGAGAAT | |
| Sequence-based reagent | EcoR1 sequence - strand | This work | 5’-ATTCTCTTGGGACCACGGTCCCAACTTGAATTCTCGGTCTGATAATCAGACTGGGACCACGGTCCCACTCGGAATTC | |
| Sequence-based reagent | 5 kb DNA primer1 | Seol and Neuman, 2011 | 5'- GCTGGGTCTCGGTTGTTCCCTTTAGTGAGGGTTAATTG | |
| Sequence-based reagent | 5 kb DNA primer2 | Seol and Neuman, 2011 | 5'- GCTGGGTCTCGTGGTTTCCCTTTAGTGAGGGTTAATTG | |
| Sequence-based reagent | DNA handle primer1 | Seol and Neuman, 2011 | 5'- GGACCTGCTTTCGTTGTGGCGTAATCATGGTCATAG | |
| Sequence-based reagent | DNA handle primer2 | Seol and Neuman, 2011 | 5'- GGGTCTCGTGGTTTATAGTCCTGTCGGGTTTC | |
| Peptide, recombinant protein | ParBF1-42 | This work | MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMAR | |
| Peptide, recombinant protein | ParBF1-42 R36A | This work | MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIAAVGVMAR | |
| Chemical compound, drug | ATP | Millipore-Sigma | A2383 | |
| Chemical compound, drug | GTP | Millipore-Sigma | G8877 | |
| Chemical compound, drug | UTP | Thermo Scientific | J23160 | |
| Chemical compound, drug | CTP | Millipore-Sigma | C1506 | |
| Chemical compound, drug | CDP | Millipore-Sigma | C9755 | 2–3% possible contamination of ParBF-CTPase substrate detected |
| Chemical compound, drug | γ32P-ATP | Perkin-Elmer | NEG002A250UC | |
| Chemical compound, drug | Dynabeads MyOne Streptavidin T1 | Invitrogen | 65601 | |
| Chemical compound, drug | Alexa Fluor 488 C5 Maleimide | Thermo Fisher | A10254 | |
| Chemical compound, drug | Alexa Fluor 594 C5 Maleimide | Thermo Fisher | A10256 | |
| Chemical compound, drug | Alexa Fluor 647 C2 Maleimide | Thermo Fisher | A20347 | |
| Chemical compound, drug | Antifoam Y-40 emulsion | Sigma | A5758 | |
| Chemical compound, drug | EDTA-free Sigmafast protease inhibitor cocktail tablet | Sigma | S8830 | |
| Chemical compound, drug | DOPC | Avanti polar lipids | 850375C | |
| Chemical compound, drug | DOPE-Biotin | Avanti polar lipids | 850149P | |
| Chemical compound, drug | Biotin-14-dCTP | Thermo Fisher | 19518018 | |
| Chemical compound, drug | Biotin-16-dUTP | Roche | 11093070910 | |
| Chemical compound, drug | Digoxigenin-11-dUTP | Roche | 11093088910 | |
| Commercial assay or kit | EnzChek Phosphate assay kit | Thermo Fisher | E6646 | |
| Software, algorithm | Prism 8 | GraphPad | Prism 8 | Used for curve fitting, and fitting parameters and their error estimation. |
| Software, algorithm | Igro Pro 7 | Wavemetrics | Igro Pro | Used for single molecule data analysis. |
| Software, algorithm | LabVIEW | National Instruments | LabView NXG Full | Used for instrumental control in single molecule experiments. |
| Software, algorithm | Metamorph 7 | Molecular Devices | Metamorph 7 | Used for TIRF michroscope data acquisition. |
| Software, algorithm | ImageJ/Fiji | National Institutes of Health | ImageJ | Used for TIRF michroscope image analysis. |
| Other (Instrument) | Prism type TIRF microscope | In house Ivanov and Mizuuchi, 2010; Vecchiarelli et al., 2013 |
Used for ParAF-ParBF complex assembly-disassembly experiments. | |
| Other (Instrument) | Magnetic tweezers | In house Seol and Neuman, 2011 and Seol et al., 2016 |
Used for taking single molecule measurements of enzyme binding on 5 kb DNA. | |
| Other (Instrument) | Plate reader | BMG Labtech | Clariostar Plus | Used for FRET-based ParBF dimerization KD and CTP hydrolysis assays using EnzChek Phosphate assay kit |