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. 2022 Oct 25;25(11):105439. doi: 10.1016/j.isci.2022.105439

Figure 2.

Figure 2

Biochemical activities of human DMC1 mutants

(A) 3D model of the human DMC1 structure with the highlighted conserved amino acids E162 (light blue) and D317 (purple). The modeling was performed based on the structure available in the protein data bank (PDB entry 4HYY).

(B) Sequence alignment of RecA family proteins. The highlighted conserved amino acids were substituted with either alanine (A) or lysine (K).

(C) Microscale thermophoresis binding curve of various DMC1 protein fractions vs calcium ion concentration (mean ± SD; n = 3).

(D) Graphical representation of DNA binding activity of DMC1 mutants in the presence of magnesium ions. Increasing concentrations of DMC1 mutants (0.1, 0.3, and 0.9 μM) were incubated with 5′-end fluorescently labelled 90-mer single-stranded DNA (pR231, 0.9-μM nucleotides), 1-mM ATP and 1-mM MgCl2 (mean ± SD; n = 3).

(E) Graphical representation of DNA binding activity of DMC1 mutants in the presence of CaCl2. Increasing concentrations of DMC1 mutants (0.1, 0.3, and 0.9 μM) were incubated with 5′-end fluorescently labelled 90-mer single-stranded DNA (pR231, 0.9-μM nucleotides), 1-mM ATP and 1-mM CaCl2 (mean ± SD; n = 3).

(F) Stability of DMC1 filaments in the presence of MgCl2 measured by bio-layer interferometry. DMC1 filaments were formed by binding DMC1 (5 μM) to 5′-biotinylated 90-mer ssDNA (1.8-μM nucleotides) pre-bound to streptavidin biosensor in the presence of 1-mM ATP and 1-mM MgCl2. The protein dissociation was measured after incubation of biosensor in a buffer containing 200-mM KCl. The data are shown as the average of three independent experiments.

(G) Stability of DMC1 filaments in the presence of calcium ion measured by BLI. DMC1 filaments were formed by binding DMC1 (5 μM) to 5′-biotinylated 90-mer ssDNA (1.8-μM nucleotides) pre-bound to streptavidin biosensor in the presence of 1-mM ATP and 1-mM CaCl2. The protein dissociation was measured after incubation of biosensor in a buffer containing 200-mM KCl. The data are shown as the average of three independent experiments. See also Figures S2 and S3.