Journal: eLife

Article Title: Sld3CBD–Cdc45 structural insights into Cdc45 recruitment for CMG complex formation during DNA replication

doi: 10.7554/eLife.101717

Figure Lengend Snippet: ( A ) Schematic of ssARS1-1–ssARS1-6. ARS1 is identified as an origin of DNA replication and divided into three 80 bp segments: ARS1-12, ARS1-34, and ARS1-56. These double-stranded DNA (dsDNA) segments could unwind into six single-stranded DNA fragments with 80 nt length: ssARS1-1, 2, 3, 4, 5, and 6. The important elements of A (autonomously replicating sequence, ARS consensus sequence), B1, B2, and B3 for unwinding are marked . We divided ssARS1-2 and ssARS1-5 fragments (blue squares) into 40-base lengths for electrophoretic mobility shift assay (EMSA). ( B ) ssDNAs were visualized using Fast Blast DNA stain on polyacrylamide gels. In the presence of ssDNA fragments, Sld3CBD, Sld3CBD–Cdc45, Sld7–Sld3ΔC–Cdc45 and Sld7–Sld3ΔC were incubated with molecular mass-related concentrations. The molecular ratio of ssDNA to protein in lanes 1, 2, 3, 4, and 5 was 1:0, 1:0.5, 1:1, 1:2, and 0:1, respectively. The controls for ssDNA and protein are lanes 1 and 5, respectively. No binding ssDNA group (ssARS1-3_1) is shown at the bottom as a negative control (NC). The overall views of the EMSA results are shown in . ( C ) The integral grayscale of the ssDNA bands was calculated and compared to the average of the ssDNA control band to determine the residual levels, showing differences in binding affinity. By three ratios, Sld3CBD-Cdc45 demonstrated a significantly ssDNA residual level (t-test, ****p<0.0001) compared to other samples, indicating low binding affinity to ssDNA. Figure 4—source data 1. PDF file containing original native-PAGE for , indicating the relevant bands and treatments. Figure 4—source data 2. Original files for native-PAGE displayed in .

Article Snippet: After electrophoresis, the reaction products were visualized using Fast Blast DNA stain (Bio-Rad Laboratories, Hercules, CA, USA) (100 × Fast Blast DNA stain diluted by 1× running buffer) or SYBR safe (Sigma-Aldrich, Burlington, MA, USA) (SYBR safe:1 × running buffer = 0.0001:1) to stain the ssDNA and Coomassie Brilliant Blue (G-250) to stain the proteins.

Techniques: Sequencing, Electrophoretic Mobility Shift Assay, Staining, Incubation, Binding Assay, Negative Control, Control, Clear Native PAGE

Journal: eLife

Article Title: Sld3CBD–Cdc45 structural insights into Cdc45 recruitment for CMG complex formation during DNA replication

doi: 10.7554/eLife.101717

Figure Lengend Snippet: ( A ) Single-stranded DNAs (ssDNAs) were visualized using Fast Blast DNA stain on polyacrylamide gels. In the presence of ssDNA fragments, Sld3CBD, Sld3CBD–Cdc45, Sld7–Sld3ΔC–Cdc45, and Sld7–Sld3ΔC were incubated with molecular mass-related concentrations. The molecular ratio of ssDNA to protein in lanes 1, 2, 3, 4, and 5 was 1:0, 1:0.5, 1:1, 1:2, and 0:1, respectively. The controls for ssDNA and protein are lanes 1 and 5, respectively. The negative controls for no binding with ssDNA (ssARS 1–3_1, NC) of each sample are shown at the bottom. The reduce or disappearance of the ssDNA band in lanes 2–4 indicates that the protein (Sld3CBD, Sld7–Sld3ΔC, and Sld7–Sld3ΔC–Cdc45) binds to ssDNA with high affinity. The smeared bands appear in high molecular weight regions of lanes 2–4, when mixed with Sld7–Sld3ΔC–Cdc45 or Sld7–Sld3ΔC, whereas no bands appeared in the negative control (NC) (ssARS1-3_1). The positions of smeared ssDNA bonds correspond to those of protein in the protein-stain pages, indicating that ssARS1 was complexed with proteins. ( B ) The proteins were visualized using Coomassie brilliant blue on gels. The EMSA experiments were conducted concurrently under equivalent conditions to ( A ). The smeared bands in the high molecular weight parts of lanes 2–4 of Sld3CBD–Cdc45, Sld7–Sld3ΔC–Cdc45, and Sld7–Sld3ΔC are shown more clearly when mixed with ssDNA. Such enhanced discernibility indicates that these proteins easily enter the gel with ssDNA, even though Sld3CBD–Cdc45 binds ssDNA weakly. Sld3CBD could not enter the gel, even when bound to ssDNA, because the pI values exceeded the pH of the running buffer (pH = 8.3). Due to limitations in protein overexpression, we utilized Sld7–Sld3ΔC–Cdc45 and Sld7–Sld3ΔC from K. marxianus (same family as S. cerevisiae ). Figure 4—figure supplement 2—source data 1. PDF file containing original native-PAGE for , indicating the relevant bands and treatments. Figure 4—figure supplement 2—source data 2. Original files for native-PAGE displayed in .

Article Snippet: After electrophoresis, the reaction products were visualized using Fast Blast DNA stain (Bio-Rad Laboratories, Hercules, CA, USA) (100 × Fast Blast DNA stain diluted by 1× running buffer) or SYBR safe (Sigma-Aldrich, Burlington, MA, USA) (SYBR safe:1 × running buffer = 0.0001:1) to stain the ssDNA and Coomassie Brilliant Blue (G-250) to stain the proteins.

Techniques: Staining, Incubation, Binding Assay, High Molecular Weight, Negative Control, Over Expression, Clear Native PAGE