Fig. 2: G4 binding protein complex MutSα stimulates DNA2-mediated G4 cleavage.

a Representative silver staining SDS-PAGE from three replicates showing the pulled-down proteins by Flag-tag M2 beads in whole cell extracts from 293T cells transfected with 3x-Flag human DNA2; b DNA replication and repair proteins co-pulled down with 3x-Flag-tagged DNA2; c, d Representative co-IP and western blot analysis from three replicates of DNA2 interaction with MSH2 and MSH6; e Representative blot from three experiments of MutSα (50 nM) and MutSβ (50 nM) incubated 2 pmol substrates/µl beads. Bead-bound MutSα (MSH2 and MSH6) or MutSβ (MSH2 and MSH3) were detected by western blot; f Representative blot from three experiments with varying concentrations of MutSα (0, 25, 50, 100, 150, 200 nM) incubated with 1 µl streptavidin magnetic bead-linked G4 substrates. Bead-bound MSH2 or MSH6 was analyzed by western blot. Binding curves: the x-axis is MSH2 (g) or MSH6 (h) concentration and the y-axis is relative intensity of MSH2 (g) or MSH6 (h). Kd was determined by sigmoid curve fitting and corresponds to the concentration at which 50% of MSH2 or MSH6 bind to G4 substrates. Concentrations from each lane in f were used to determine the binding affinity of MSH2 (48 nM) and MSH6 (43 nM) to G4B oligo; i AIRYSCAN confocal microscopy of G4s; MSH2-G4 co-immunofluorescence staining in MEFs (top panel). The bottom panel shows identified spots of MSH2 and G4. Scale bar = 1 μm; j Frequency of co-localization of MSH2 and G4. Red line indicates mean ± SEM. P-value was calculated using a two-sided paired t-test (n = 79 cells, p < 0.0001); k The representative denaturing PAGE image shows DNA2 (16 nM) cleaving the ³²P-labeled G4 substrate (top) in the absence or presence of MutSα (50 nM) or XPE (100 nM); l The representative denaturing PAGE image shows cleavage of the ³²P-labeled G4 bubble substrate (top) by the DNA2 (16 nM) in the absence or presence of MutSα (50 nM) or MutSβ (50 nM). Source data are provided as a Source data file. Created in BioRender. Zhou, T. (2025) https://BioRender.com/2d65a6r.