Journal: Nucleic Acids Research

Article Title: The ATPase activity of yeast chromosome axis protein Hop1 affects the frequency of meiotic crossovers

doi: 10.1093/nar/gkae1264

Figure Lengend Snippet: Purification, SDS-PAGE analysis and thermal stability of WT Hop1 and its variants carrying amino acid substitutions in the putative ATP binding site. ( A ) Schematic diagram of Hop1 domain structure: an N-terminal HORMA domain, and a C-terminal domain (Hop1-CTD) including the zinc finger motif. The aa residues potentially involved in ATP binding/hydrolysis are indicated below the linear schematic diagram. ( B ) Shown is a Coomassie blue-stained SDS-PAGE gel showing the homogeneity of purified WT and Hop1 variants. Lane 1, molecular weight standards. Lanes 2–9, SDS-PAGE analysis of purified WT Hop1 and its variants (5 μg protein in each lane). ( C ) Western blot analysis of WT Hop1 and its variants using anti-Hop1 antibodies. The closed arrowheads in panels (B) and (C) denote the Hop1 degradation product. ( D ) A Coomassie blue-stained SDS-PAGE gel of protein samples from various stages of Hop1-CTD purification. Lane 1, molecular weight standards; lane 2, whole-cell lysate from uninduced cells (25 μg protein); lane 3, whole-cell lysate from induced cells (25 μg protein); lane 4, eluate from Ni 2+ -NTA affinity column (4 μg protein); lane 5, eluate from Superdex 200 column (4 μg protein). ( E ) A Coomassie blue-stained SDS-PAGE gel of protein samples from various stages of purification of Hop1 HORMA domain. Lane 1, molecular weight standards; lane 2, whole-cell lysate from uninduced cells (30 μg protein); lane 3, whole-cell lysate from induced cells (30 μg protein); lane 4, eluate from Ni 2+ -NTA affinity column (5 μg). ( F ) Thermal denaturation profiling of WT Hop1 and its variants. ( G ) T m values of WT Hop1 and its variants. The data are presented as the mean ± SD from three independent experiments.

Article Snippet: The Ni 2+ -NTA resin, Sepharose G-50, isopropyl-β-D-thiogalactopyranoside (IPTG) and molecular weight standards were purchased from G-Biosciences (St. Louis, MO, USA).

Techniques: Purification, SDS Page, Binding Assay, Staining, Molecular Weight, Western Blot, Affinity Column

Journal: Nucleic Acids Research

Article Title: The ATPase activity of yeast chromosome axis protein Hop1 affects the frequency of meiotic crossovers

doi: 10.1093/nar/gkae1264

Figure Lengend Snippet: Hop1 has intrinsic ATPase activity. ( A ) A Coomassie blue-stained SDS-PAGE gel showing analysis of protein samples at different stages of purification of Hop1. Lane 1, molecular weight standards; lane 2, whole-cell lysate from uninduced cells (25 μg); lane 3, whole-cell lysate from induced cell lysates (25 μg); lane 4, eluate from Ni 2+ -NTA affinity column (5 μg); lane 5, eluate from Superdex 200 column (3 μg); lane 6, western blot analysis of purified Hop1 (fraction 5) using anti-Hop1 antibodies. ( B ) A thin-layer chromatogram showing [γ- 32 P]ATP hydrolysis by Hop1 in a concentration-dependent manner. Increasing concentrations of Hop1 were incubated with 20 μM cold ATP (and 200 pM [γ- 32 P]ATP as a tracer) at 37°C for 40 min. The reaction products were analyzed by TLC. ( C ) Graph shows quantification of the ATPase activity ( n = 3). ( D ) Hop1 binds to [α- 32 P]ATP. The reaction mixtures containing indicated proteins were incubated with 0.5 nM [α- 32 P]ATP. Samples were analyzed using a nitrocellulose filter binding assay. Lane 1, S. cerevisiae Rev7 (1 μg); lane 2, No protein (control). lanes 3–7, various concentrations of Hop1 (0.2, 0.4, 0.8, 1.2 and 1.6 μg), and lane 8, MtRecA (0.4 μg). ( E ) The graph shows the quantification of [α- 32 P]ATP-cross-linked species ( n = 3). ( F ) Purification of Hop1 expressed in the cell-free translation system. Samples at various stages of purification of Hop1 were analyzed by SDS-PAGE and staining with Coomassie blue. Lane 1, molecular weight standards; lane 2, 5 μg protein of the translation mixture (input); lane 3, Ni 2+ -NTA column flow-through (5 μg); lane 4, column wash with 20 mM imidazole (5 μg). Lanes 5–6, protein eluted with 150 and 250 mM imidazole. Lane 7, elution with 500 mM imidazole (Imid). ( G ) A thin-layer chromatogram showing Hop1 made in the cell-free protein synthesis system catalyses [γ- 32 P]ATP hydrolysis. Various concentrations of Hop1 (0.1–1 μM) were incubated with 20 μΜ cold ATP (and 200 pM [γ- 32 P]ATP as a tracer) for 30 min at 37°C. ( H ) A thin-layer chromatogram showing time course of ATP hydrolysis by Hop1 from the cell-free protein synthesis system. Lane1, reaction performed in the absence of Hop1. Lanes 2–8 correspond to increasing reaction times as follows: 5, 10, 15, 20, 30, 45 and 60 min, respectively. The reaction products were analyzed as in panel (B). (B, G and H) 2 μl from each reaction mixture was spotted on a TLC plate and developed in a solution containing 1 M HCOOH, 0.5 M LiCl and 1 mM EDTA. The TLC plates were dried and imaged using a Fuji FLA-9000 phosphorimager.

Article Snippet: The Ni 2+ -NTA resin, Sepharose G-50, isopropyl-β-D-thiogalactopyranoside (IPTG) and molecular weight standards were purchased from G-Biosciences (St. Louis, MO, USA).

Techniques: Activity Assay, Staining, SDS Page, Purification, Molecular Weight, Affinity Column, Western Blot, Concentration Assay, Incubation, Filter-binding Assay, Control