Figure 4.

Impact of Polµ DNA-binding properties on its enzymatic activity during NHEJ. (A) Gap-filling activity of Polβ, Polλ and Polµ (25 nM each) was assayed using a substrate formed by the hybridization of the oligonucleotides SP1C, T28 and D12. When indicated, 10 nM of each dNTP was added, in the presence of 2.5 mM MgCl₂. (B) NHEJ assay of Polβ (600 nM), Polλ (600 nM) and Polµ (200 nM) was performed as described in ‘Materials and Methods’ section, using a set of compatible substrates: the labeled substrate was formed by hybridization of GT and NHEJ-D (shown in light gray) and the cold substrate by hybridization of CA and NHEJ-D (shown in dark gray). When indicated, dNTPs were added separately at 100 µM in the presence of 1 mM MnCl₂ for Polβ and Polλ, and 2.5 mM MgCl₂ for Polµ. After electrophoresis, the labeled fragments were detected by autoradiography. (C) NHEJ reaction performed as in (B), with a set of incompatible substrates in which both the labeled (light gray) and cold (dark gray) molecules were formed by hybridization of C- and D-NHEJ. When indicated, the substrates contain a 5′-P group at the downstream strand (dark gray spheres).