Figure 1.

Loop1 confers Polμ its NHEJ efficiency on short 3′-overhangs. (A) EMSA was performed for the wild-type and mutant Polμ at the indicated amounts (0.5, 1, 2 μM) using a 3′-protruding substrate (1 nM) containing oligonucleotides 1TG and 1D-NHEJ. When indicated, 2.5-mM MgCl₂ and/or 10-μM dGTP were added. After electrophoresis, gel was dried and the labelled fragments were detected by autoradiography. (B) NHEJ reactions were performed with 200 nM of the indicated proteins using four sets of substrates: the labelled substrates were formed by hybridization of 1TTTG, 1TTG, 1TG or 1C with 1D-NHEJ, and the cold substrates by hybridization of either 1AAAC, 1AAC, 1AC or 1C with 1D-NHEJ. The grey spheres indicate the presence of a 5′-P group in the downstream strand of the substrate. When indicated, each of the four ddNTPs (10 μM) was added in the presence of 2.5-mM MgCl₂ or 1-mM MnCl₂.