Figure 4.

Residues Contacting the Template Strand: Implications for PolDom-Mediated NHEJ Reactions

(A) EMSAs were performed for the indicated proteins (200 nM) using a gapped substrate containing the oligonucleotides SP1C, T13C, and DG-P. When indicated, 1 mM MnCl₂ and/or 100 μM UTP was added. After electrophoresis, the gel was dried and the labeled fragments were detected by autoradiography.

(B) Footprinting assays of wild-type or mutant PolDom (5 μg) were conducted as described in the Experimental Procedures. BSA (10 μg) was added to the control lane. The substrate was formed with oligonucleotides FP-T, FP-P, and FP-D, depicted on the right.

(C) Gap-filling reactions were performed as described in Experimental Procedures for the indicated proteins (25 nM) using a gapped DNA substrate containing the oligonucleotides SP1C, T13C, and DG-P. When indicated, NTPs were added separately at 10 nM in the presence of 1 mM MnCl₂.

(D) NHEJ reactions were performed with 600 nM of the indicated proteins using a set of DNA substrates formed with the oligonucleotides TG with NHEJ-D and AC with NHEJ-D2. When indicated, each of the four NTPs (100 μM) was added in the presence of 1 mM MnCl₂.

See also Figure S4.