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. 2021 Dec 30;298(2):101552. doi: 10.1016/j.jbc.2021.101552

Figure 7.

Figure 7

Mutant alpha-synuclein and beta- and gamma-synuclein do not bind DNA like wild-type alpha-synuclein.A1, left, increasing concentrations of wild-type (WT) alpha-synuclein (aSyn) and disease-causing point mutations (A30P, E46K, A53T) shift 300 bp DNA to higher apparent lengths, with A30P and A53T aSyn being less efficient at shifting than WT, and E46K being more efficient and shifting to a higher apparent length than WT. Right, deletion of the central aggregation-prone NAC domain of aSyn (deltaNAC) increases the efficiency of shifting but reduces the apparent length of the shifted species compared to WT. A2, Coomassie stain showing aSyn proteins loaded into each lane. A3, DNA gel (green) and Coomassie stain (red) localization from the same experiment. A4, group data showing shifted fraction as a function of aSyn concentration (aSyn R2: WT = 0.980, A30P = 0.963, E46K = 0.999, A53T = 0.965; shifted fraction 1.4 μM aSyn: WT = 0.550 ± 0.082, A30P = 0.270 ± 0.047, E46K = 0.975 ± 0.028, A53T = 0.379 ± 0.102; shifted fraction 2.9 μM aSyn: WT = 0.743 ± 0.049, A30P = 0.515 ± 0.074, E46K = 0.996 ± 0.007, A53T = 0.611 ± 0.010; at 1.4 μM one-way ANOVA p < 0.0001; post-hoc Tukey tests WT versus A30P, E46K, A53T all p between 0.0002 and 0.0449; at 2.9 μM one-way ANOVA p < 0.0001; post-hoc Tukey tests WT versus A30P, E46K, A53T all p between 0.0004 and 0.0174; shifted fraction 1.4 μM aSyn: WT = 0.597 ± 0.148, deltaNAC = 1.000 ± 0.000; shifted fraction 2.9 μM aSyn: WT = 0.764 ± 0.113, deltaNAC = 1.000 ± 0.000; at 1.4 μM t test p = 0.0092; at 2.9 μM t test p = 0.0225; three-parameter dose–response curve; N = 3 gels, x-axis aSyn concentrations on log scale). B1, increasing aSyn concentration produces a shift of 300 bp DNA to an apparent length of ∼600 bp, while beta-synuclein (bSyn) and gamma-synuclein (gSyn) produce no such shift. B2, Coomassie stain showing synuclein proteins loaded into each lane. B3, DNA gel (green) and Coomassie stain (red) localization from the same experiment. B4, group data showing shifted fraction as a function of synuclein concentration (aSyn R2 = 0.791; shifted fraction: 29 μM aSyn = 0.039 ± 0.020, 57 μM aSyn = 0.199 ± 0.066, all other values including for all bSyn and gSyn concentrations produced an undetectable shift = 0.0; at 29 μM one-way ANOVA p = 0.0087; post-hoc Tukey test aSyn versus bSyn, and aSyn versus gSyn p = 0.0139; at 57 μM one-way ANOVA p = 0.0010; post-hoc Tukey test aSyn versus bSyn, and aSyn versus gSyn p = 0.0017; three-parameter dose–response curve; N = 3 gels, x-axis Syn concentrations on log scale).