Figure 3.
Key Flips have impacts on the binding of ALKBH6 to ssDNA.A, key loops of ALKBH6. B, overlay analysis of ALKBH6, ALKBH2, and ALKBH3 around the Flip domains; secondary structures are labeled based on ALKBH6 (ALKBH6, cyan; ALKBH2, yellow, PDB code 3S57; ALKBH3, warm pink, PDB code 2IUW). C, the binding of His-tag labeled ALKBH6 with different unlabeled ssDNAs, KD measured by MST. D, EMSA assay shows the binding of ALKBH6 with FAM-labeled ssDNA1. The EMSA assay was detected by the fluorescence of FAM. Unlabeled ssDNA1 is a competitor of the labeled ssDNA1 to rule out spurious interactions. E, ALKBH6 binding with different FAM-labeled bubble DNAs. The experiments were repeated three times. F, structure-based sequence alignment of ALKBH6 in different species or its family members within the Flip3 region. Secondary structural elements are represented according to the structure of ALKBH6. hALKBH6, human ALKBH6; mALKBH6, mouse ALKBH6 (Q8K2U2); dALKBH6, Danio rerio ALKBH6 (Q6IQE9); xALKBH6, Xenopus laevis (Q5PQ59). Cylinders and arrows represent α-helices and β-sheets, respectively. Invariable residues are highlighted. The length of sequences omitted for the clarity of the presentation are shown in brackets. Evidently, Flip3 in red font shaded yellow is a unique motif of human ALKBH6.