Figure 3.

Mutation in the ligase I gene results in decreased MHEJ. (A) Western blot analysis with the antibody against DNA ligase I. GM16096 cells are primary human fibroblast cells that carry two different missense mutations in the ligase I gene. WI38 cells are primary normal human fibroblasts. (B) End-joining activities. End-joining assays were carried out using 1 μg nuclear extract prepared from GM16096 or WI38 fibroblast cells. (C) Quantification of MHEJ activity. (D) Inhibition of end-joining by anti-Ligase 1 antibodies. Nuclear extract from HTD114 cells (1.6 µg total protein) was incubated for 30 min on ice with 1 µg antibody against human DNA ligase I, then incubated with 400 ng linear DNA at 14°C. After 1 h incubation, DNA products were analyzed by agarose gel electrophoresis. (E) Quantification of MHEJ affected by anti-ligase 1 antibodies.