Figure 4.

Mutation of the interacting thumb residue Arg⁴⁴⁶ mimics disruption of the brooch. (A) Multiple sequence alignment showing the region corresponding to helix N in the four X family polymerases from different species. Numbers indicate the residues in the human Polµ sequence. The four conserved arginines (Arg⁴⁴⁴, Arg⁴⁴⁷, Arg⁴⁴⁸ and Arg⁴⁵¹ in mouse Polµ) oriented towards the negatively charged phosphate backbone of the template strand are boxed in grey or black and indicated with an asterisk. Arg⁴⁴⁶ in human Polµ is numbered. (B) NHEJ reactions were performed for 30 min at 30°C with 400 nM of the indicated proteins and using two sets of substrates: the labelled substrate contained 1TG and 1D-NHEJ and the cold one 2AG and 2D-NHEJ. Each of the four ddNTPs (100 µM) was added in the presence of 2.5 mM MgCl₂. (C) NHEJ reactions were performed as in (B) but using non-complementary DNA substrates, the labelled one containing 1C and 1D-NHEJ, and the cold one containing 1C and 2D-NHEJ. (D) Gap-filling reactions were performed for 30 min at 30°C with the indicated proteins (100 nM) using a gapped substrate containing SP1C, T13C and DG1-P. dGTP was added separately at the indicated concentrations in the presence of 2.5 mM MgCl₂.