proteins high q support  (Bio-Rad)

Journal: Nucleic Acids Research

Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

doi: 10.1093/nar/gks1088

Figure Lengend Snippet: Identification of specific protein complexes of Pif1 in S. cerevisiae using I-DIRT. ( A ) Schematic representation of the I-DIRT procedure. Pif1-TAP tag strains were cultured in a light isotopic media, whereas the parent strains were cultured in heavy isotopic media containing d4-lysine. Equal quantities of cell lysates were mixed and subjected to affinity capture for the Pif1-TAP protein complex followed by SDS–PAGE and MS analysis. ( B ) Immunoisolated Pif1-TAP and its associated proteins were resolved by SDS–PAGE on a 4–20% NuPAGE gel and visualized by Coomassie blue staining. ( C ) Representative mass spectra of peptides from the Pif1 I-DIRT experiment. A peptide from the Ssb2 protein containing a single lysine exhibits both isotopically light (1394.8 Da) and heavy (1398.8 Da) peptides. The Rim1 peptide has two lysines with the monoisotopic peak exhibiting only isotopically light peptides (1448.76 Da). As expected, the peptides of Pif1 protein exhibited only the monoisotopic peak for isotopically light peptides. ( D ) For each of the lysine containing peptides identified by MS, the peak area under the isotopically light peptides was compared with the peak area for the heavy peptides to obtain a ‘fraction light’. Twenty two proteins were identified as nonspecific interactors due to one or more peptides having a light to heavy ratio of ∼0.6. In contrast, the fraction of light to heavy peptides for Rim1 and Pif1 proteins was ∼1. All the identified proteins and their average ‘fraction light’ areas are listed in Supplementary Table S1 .

Article Snippet: Rim1 protein was further purified by passing it through a strong anion exchange column (Macro-Prep High Q Support, Bio-Rad) with a gradient salt elution of 150 mM to 2 M NaCl.

Techniques: Cell Culture, SDS Page, Staining

Journal: Nucleic Acids Research

Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

doi: 10.1093/nar/gks1088

Figure Lengend Snippet: Purification and characterization of recombinant Rim1 protein and its C-terminal truncation variant. ( A ) Multiple sequence alignment of eukaryotic mitochondrial SSBs and bacterial SSBs using the ClustalW2 program to determine the C-terminal tail region of Rim1. The sequences for H. sapiens mtSSB ( Hs mtSSB) (GenBank™ accession: NP_003134), Xenopus laevis mtSSB ( Xl mtSSB) (GenBank™ accession: NP_001095241), Bombyx mori mtSSB ( Bm mtSSB) (GenBank™ accession: ABF51293), D. melanogaster mtSSB ( Dm mtSSB) (GenBank™ accession: AAF16936), E. coli SSB ( Ec SSB) (GenBank™ accession: YP_859663), Thermotoga maritima ( Tm SSB) (GenBank™ accession: Q9WZ73), Deinococcus radiodurans SSB ( Dr SSB) (GenBank™ accession: Q9RY51) and S. cerevisiae Rim1 ( Sc Rim1) (GenBank™ accession: AAB22978) are used for the alignment. The sequence alignment determined that the first 100 amino acids from the N-terminal end of Rim1 are involved in formation of the OB-fold domain, and the remaining 18 amino acids from the C-terminal end form the putative unstructured tail region. The amino acid sequences involved in the formation of the C-terminal tails of SSB proteins are highlighted in gray. The C-terminal tail of Rim1 contains five acidic amino acids that are indicated in bold. ( B ) Coomassie blue stained 15% SDS–PAGE gel to visualize purified Rim1 (lane 2) and Rim1ΔC18 (lane 2). The purified proteins were >95% homogenous as assessed from the gel. ( C ) SEC-MALS detection reveals that the Rim1 and Rim1ΔC18 exist as a tetramer. The theoretical MM of monomeric Rim1 and Rim1ΔC18 is 13.29 and 11.43 kDa, respectively. The observed MM and hydrodynamic radius ( R h ) for Rim1 and Rim1ΔC18 proteins are as indicated. ( D ) Rim1 binding affinity for ssDNA was evaluated by fluorescence anisotropy. The anisotropy values for Rim1 binding to 1 nM 3′F-T 20 (open diamonds) and 3′F-T 70 (closed diamonds) were plotted as average values from three experiments with a standard deviation. Rim1 binding data to 3′F-T 20 were fit to the Hill equation resulting in a Hill coefficient of 2.5 and an apparent K d of 3.1 ± 0.1 nM (tetramer). Rim1 binding to 3′F-T 70 is stoichiometric under the conditions used here ( K d value <1 nM). ( E ) Anisotropy values for binding of Rim1ΔC18 to 1 nM 3′F-T 20 (open triangles) and 3′F-T 70 (closed triangles) were plotted as averages from three experiments with a standard deviation. Rim1ΔC18 binding to 3′F-T 20 was fit to the Hill equation resulting in a Hill coefficient value of 1.6 ± 0.1 and an apparent K d value of 3.5 ± 0.2 nM (tetramer). Rim1ΔC18 binding to 3′F-T 70 resulted in two apparent binding modes. A tight binding mode that appears similar to Rim1 ( K d value <1 nM) and a weaker binding mode that did not saturate under these conditions.

Article Snippet: Rim1 protein was further purified by passing it through a strong anion exchange column (Macro-Prep High Q Support, Bio-Rad) with a gradient salt elution of 150 mM to 2 M NaCl.

Techniques: Purification, Recombinant, Variant Assay, Sequencing, Staining, SDS Page, Binding Assay, Fluorescence, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

doi: 10.1093/nar/gks1088

Figure Lengend Snippet: In vitro co-precipitation experiments reveal a direct interaction between Rim1 and Pif1 proteins and two possible sites of interactions on Rim1. ( A ) Ammonium sulfate co-precipitation of Pif1 with Rim1 or Rim1ΔC18. The presence of Pif1, Rim1, Rim1ΔC18 and 270 g/l ammonium sulfate in the reaction are indicated by plus symbols. Both pellet and supernatant fractions were analyzed on a 15% SDS–PAGE gel. Rim1 alone (lane 3) or Rim1ΔC18 alone (lane 7) precipitate very little in the presence of ammonium sulfate; however, they co-precipitate completely with Pif1 under the same conditions (lane 5 and 9, respectively). ( B ) Co-precipitation of Pif1 protein with SSB-coated Dynabeads was performed as described in ‘Materials and Methods’ section. Purified Rim1, Rim1ΔC18 or Hs mtSSB protein was coated onto epoxy activated Dynabeads. As a negative control, Dynabeads were coated with glycine or BSA. SSB coated Dynabeads were incubated with equal amounts of purified Pif1. Dynabeads were captured with a magnet, washed and proteins were eluted using SDS–PAGE loading buffer followed by separation on a 4–20% resolving gel. Pif1 did not co-precipitate with glycine-coated (lane 2) or BSA-coated (lane 3) Dynabeads. Pif1 co-precipitated with Rim1-coated beads (lane 4) and its association was not affected in the presence of DNase I (lane 5). Pif1 was also observed to co-precipitate with Hs mtSSB-coated (lane 6) and Rim1ΔC18-coated Dynabeads (lane 7). ( C ) A semi-quantitative measurement of relative Pif1 protein association with different SSB-coated Dynabeads from (B). Pif1 protein co-precipitated with each SSB-coated Dynabead was quantified using ImageQuant software and normalized to the amount of SSB protein on the gel. Pif1 association with Rim1-coated beads was taken as 1 and the relative amount of Pif1 co-precipitated with Hs mtSSB or Rim1ΔC18-coated beads was 0.63 and 0.45, respectively.

Article Snippet: Rim1 protein was further purified by passing it through a strong anion exchange column (Macro-Prep High Q Support, Bio-Rad) with a gradient salt elution of 150 mM to 2 M NaCl.

Techniques: In Vitro, SDS Page, Purification, Negative Control, Incubation, Software

Journal: Nucleic Acids Research

Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

doi: 10.1093/nar/gks1088

Figure Lengend Snippet: The OB-fold domain and C-terminal tail of Rim1 form two independent Pif1 interaction sites. ( A ) Schematic diagram of the procedure used to measure the binding affinity between Pif1 and SSB protein. Rim1 or Rim1ΔC18 was labeled with the amine reactive fluorescein dye 5-FAM SE as described in ‘Materials and Methods’ section. The labeled proteins were used in binding assays to measure the change in fluorescence anisotropy as a function of protein binding. ( B ) FAM-labeled Rim1 protein binds to unlabeled Rim1 as indicated by increasing anisotropy; however, it did not bind to Hs mtSSB. FAM-labeled Rim1ΔC18 also did not bind to Hs mtSSB. ( C ) FAM-labeled Rim1 or FAM-labeled Rim1ΔC18 was titrated with Pif1 protein. The average anisotropy values from at least three independent experiments with a standard deviation were plotted using KaleidaGraph and fit to the equation for a hyperbola to obtain dissociation constants ( K d ) of 0.69 ± 0.03 and 2.5 ± 0.6 µM for Pif1 interaction with FAM-Rim1 and FAM-Rim1ΔC18, respectively.

Article Snippet: Rim1 protein was further purified by passing it through a strong anion exchange column (Macro-Prep High Q Support, Bio-Rad) with a gradient salt elution of 150 mM to 2 M NaCl.

Techniques: Binding Assay, Labeling, Fluorescence, Protein Binding, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

doi: 10.1093/nar/gks1088

Figure Lengend Snippet: Rim1 and Rim1ΔC18 stimulate Pif1 DNA helicase activity. ( A ) Pif1-catalyzed separation of a partial duplex DNA substrate, 70T30bp, under multiple turnover conditions in the presence or absence of Rim1 or Rim1ΔC18 protein. ( B ) Formation of ssDNA product over time was quantified and plotted as the average of at least three independent reactions with a standard deviation for Pif1 alone (circles), Rim1+Pif1 (squares) and Rim1ΔC18+Pif1 (triangles) from (A). The data were fit to a single exponential resulting in observed rate constants of 0.44 ± 0.04, 1.8 ± 0.1 and 0.90 ± 0.02 per min for Pif1 alone, Rim1+Pif1 and Rim1ΔC18+Pif1, respectively. ( C ) Pif1-catalyzed separation of a partial duplex DNA substrate, 20T30bp, under multiple turnover conditions in the presence or absence of Rim1 or Rim1ΔC18 protein. ( D ) The fraction of ssDNA product formed over time for Pif1 alone (circles), Rim1+Pif1 (squares) and Rim1ΔC18+Pif1 (triangles) from (C) was quantified and plotted as the average of at least three independent reactions with a standard deviation. The observed rate constants for Pif1 alone, Rim1+Pif1 and Rim1ΔC18+Pif1 were 0.30 ± 0.01, 0.31 ± 0.01 and 0.25 ± 0.01 per min, respectively.

Article Snippet: Rim1 protein was further purified by passing it through a strong anion exchange column (Macro-Prep High Q Support, Bio-Rad) with a gradient salt elution of 150 mM to 2 M NaCl.

Techniques: Activity Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

doi: 10.1093/nar/gks1088

Figure Lengend Snippet: Effect of heterologous SSBs on Pif1-catalyzed DNA helicase activity. ( A ) The fraction of ssDNA product formed under multiple turnover conditions with the 70T30bp substrate for Hs mtSSB+Pif1 (triangles), and gp32+Pif1 (diamonds) was plotted as the average value of three independent experiments along with Pif1 alone (circles) and Rim1+Pif1 (squares) which is replotted for comparison from B. The data were fit to a single exponential resulting in observed rate constants for product formation of 0.44 ± 0.04, 1.84 ± 0.09, 1.1 ± 0.1 and 0.58 ± 0.02 per min for Pif1 alone, Rim1+Pif1, Hs mtSSB+Pif1 and gp32+Pif1, respectively. ( B ) Results of DNA strand separation experiments conducted with the 20T30bp substrate. The fraction of ssDNA formed over time for Pif1 alone (circles), Rim1+Pif1 (squares), Hs mtSSB+Pif1 (triangles) and gp32+Pif1 (diamonds) was plotted as the average of at least three independent experiments. The observed rate constants for Pif1 alone, Rim1+Pif1, Hs mtSSB+Pif1 and gp32+Pif1 were 0.30 ± 0.01, 0.31 ± 0.01, 0.26 ± 0.02 and 0.25 ± 0.01 per min, respectively.

Article Snippet: Rim1 protein was further purified by passing it through a strong anion exchange column (Macro-Prep High Q Support, Bio-Rad) with a gradient salt elution of 150 mM to 2 M NaCl.

Techniques: Activity Assay, Comparison

Journal: Nucleic Acids Research

Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

doi: 10.1093/nar/gks1088

Figure Lengend Snippet: The N-terminal domain of Pif1 is essential for Rim1 mediated stimulation of helicase activity. ( A ) Schematic diagram of the Pif1 variants used: the N-terminal deletion mutant (Pif1ΔN) and the C-terminal deletion mutant (Pif1ΔC). ( B ) Results of Pif1ΔN-catalyzed separation of a partial duplex DNA substrate, 70T30bp, under multiple turnover conditions in the presence or absence of Rim1. The fraction of ssDNA formed over time for Pif1ΔN (closed squares) and Pif1ΔN+Rim1 (open diamonds) was plotted as the average of at least three independent experiments. The observed rate constants for Pif1ΔN and Pif1ΔN+Rim1 were 0.52 ± 0.03 and 0.46 ± 0.03 per min, respectively. ( C ) Binding affinity of Pif1ΔN with FAM-Rim1. Fluorescence anisotropy of FAM-Rim1 was plotted as a function of increasing concentrations of Pif1ΔN. Data were fit to the equation for a hyperbola to obtain a K d value of 1.6 ± 0.2 µM.

Article Snippet: Rim1 protein was further purified by passing it through a strong anion exchange column (Macro-Prep High Q Support, Bio-Rad) with a gradient salt elution of 150 mM to 2 M NaCl.

Techniques: Activity Assay, Mutagenesis, Binding Assay, Fluorescence

Journal: Nucleic Acids Research

Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

doi: 10.1093/nar/gks1088

Figure Lengend Snippet: Rim1 has no effect on the k cat value for ATP hydrolysis catalyzed by Pif1. ( A ) DNA stimulated ATPase activity of Pif1 (100 nM) in the presence or absence of Rim1 (100 nM) at increasing concentrations of poly(dT). The ATPase activity of Pif1 was plotted as the average value from three independent experiments and data were fit to a hyperbola to obtain kinetic constants k cat and K eff . The observed k cat value for Pif1 was 94.7 ± 4.9 per s and it did not change in the presence of Rim1 (96.7 ± 1.8 per s). The measured K eff value for Pif1 was 1.07 ± 0.2 µM and it increased by 3-fold in the presence of Rim1 (3.4 ± 0.2 µM). ( B ) DNA-stimulated Pif1 (20 nM) ATPase activity at saturating concentrations of poly(dT) (20 µM) was measured with increasing concentrations of Rim1. The average Pif1 ATPase activity from three independent experiments was plotted. Titration with Rim1 had no effect on Pif1 ATPase activity.

Article Snippet: Rim1 protein was further purified by passing it through a strong anion exchange column (Macro-Prep High Q Support, Bio-Rad) with a gradient salt elution of 150 mM to 2 M NaCl.

Techniques: Activity Assay, Titration