λ dna tethering step  (Thermo Fisher)

Journal: bioRxiv

Article Title: Mcm10 and RecQL4 Synergize to Activate the Eukaryotic Replicative DNA Helicase

doi: 10.1101/2025.08.05.668837

Figure Lengend Snippet: (a) Domain diagram for frog (x), human (h), and mouse (m) RecQL4 compared to yeast (y) Sld2. ATPase (blue) and R4ZBD (green) domain boundaries for human RecQL4 are taken from a structural study the Kisker lab and extrapolated to its orthologs using multiple-sequence alignment (see ). Sld2-like region boundaries (yellow) are defined here based on the multiple-sequence alignment and serve as a rough indicator of sequence conservation. C-terminal lobe boundaries (orange) were defined based on AlphaFold3 predictions and multiple-sequence alignment (see ). (b) Diagram illustrating the workflow of the ensemble plasmid replication assay (dCTP P32 is added to the reaction and is incorporated into nascent DNA). (c) Autoradiograph of agarose gel showing the results of a plasmid replication assay performed in mock-depleted, RecQL4-depleted, or Mcm10-and- RecQL4-depleted extract. Reactions were supplemented with buffer, 100 nM of recombinant Mcm10 expressed in insect cells (Mcm10 Sf9 ), or 100 nM of Mcm10 expressed using In Vitro Transcription-Translation (Mcm10 IVTT ). Time-points were collected at 10, 20, 30, and 60 minutes. (d) Quantification of the nascent DNA signal for each reaction shown in panel (c) . (e) Immunoblots of extract samples taken from reactions shown in panel (c) . Both Mcm10 and RecQL4 depletions are very stringent (less than 1% of endogenous protein remaining). GINS serves as a loading control. Double-depleted extract does not support any DNA replication. Both Mcm10 Sf9 and Mcm10 IVTT support similar levels of DNA replication as RecQL4-depleted extract, indicating that the recombinant protein preparations have similar biochemical activity as the endogenous Mcm10. (f) Representative kymograms illustrating Mcm10 AF647 binding to licensed DNA in mock-depleted or DONSON-depleted extract. To calculate the Mcm10 binding time on DNA, the durations of all binding events are added, and normalized by the length of the DNA molecule. This is done for several stretched λ DNA molecules (typically 100-200 DNA molecules). This measurement is corrected for non-specific binding of Mcm10 AF647 off the DNA by performing a similar analysis in regions of interest that contain no DNA molecules, as previously described .

Article Snippet: In the λ DNA tethering step, 20 pM of streptavidin AF647 (Thermo Fisher Scientific, Cat# S21374) was added to the streptavidin solution.

Techniques: Sequencing, Plasmid Preparation, Autoradiography, Agarose Gel Electrophoresis, Recombinant, In Vitro, Western Blot, Control, Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: Mcm10 and RecQL4 Synergize to Activate the Eukaryotic Replicative DNA Helicase

doi: 10.1101/2025.08.05.668837

Figure Lengend Snippet: (a) Representative kymograms illustrating RecQL4 AF647 (orange) binding to licensed DNA. Fen1 mKikGR (blue) marks nascent DNA. (b) Quantification of RecQL4 AF647 binding to DNA. Total DNA length analyzed is reported in parentheses. (c) Fraction of productive (green) versus non-productive (gray) RecQL4 AF647 binding events. N – number of binding events analyzed. (d) Number of origins fired per DNA molecule. N – number of λ DNA molecules analyzed. (e) Time delay between the start of the experiment (addition of nucleoplasmic extract) and origin firing. N – number of origin firing events. Blue bars and gray boxes denote the median and 95% CI. In (b) , (c) , (d) error-bars denote the 95% CI. Two-sample Kolmogorov-Smirnov test was used to compute p-values: ns (not significant, p > 0.05), * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001).

Article Snippet: In the λ DNA tethering step, 20 pM of streptavidin AF647 (Thermo Fisher Scientific, Cat# S21374) was added to the streptavidin solution.

Techniques: Binding Assay