Figure 4. Functionality of the MCM4 isoforms detected in the cells of the patient.

(A) The Triton X–extractable fraction from patient and control SV40 fibroblasts was subjected to immunoprecipitation with a monoclonal antibody against MCM2. Protein extracts and immunoprecipitates were analyzed by immunoblotting with antibodies against MCM4, MCM3, MCM5, and MCM6. MCM2 was used as a loading control. (B) The chromatin-bound fraction was subjected to immunoprecipitation with a monoclonal antibody against MCM2. This procedure was carried out on the DNase I–extracted fractions of both control cells and cells from the patient. Protein extracts and immunoprecipitates were analyzed by immunoblotting with antibodies against MCM4, MCM3, MCM5, and MCM6. MCM2 was used as a loading control. Immunoprecipitation with IgG was used as a negative control. (C) Representative flow cytometry plots of the cell cycle of SV40 fibroblasts from controls and patients. Control (left), P1.3 (middle), and P2.1 (right) cell cycles in the absence of treatment. Transformation of cells with SV40 T antigen causes increased ploidy of all cells (59). P1 corresponds to normal G₁ phase, P2+P3+P4 correspond to normal S phase, P5 corresponds to normal G₂ phase plus abnormal G₁ or failed mitosis, P6+P7 correspond to re-replication S phase, and P8 corresponds to 8C (P8) DNA content. Patients’ SV40 fibroblasts with or without 0.3 μM aphidicolin (Aph) treatment. (D) Representative chromosome spreads of chromosome breaks induced with or without aphidicolin. A WT metaphase chromosome without aberrations (left); a P1.3 metaphase with some aberrations indicated by blue arrowhead (middle); and a P1.3 metaphase after aphidicolin treatment, with chromosomal aberrations indicated by arrowheads (right). Blue arrowheads indicate chromatid breaks, and red arrowheads indicate chromosome exchanges. Bottom: Chromosome breaks (mean) per metaphase in P1.3 and P2.1 SV40 fibroblasts and in control SV40 fibroblasts. Complementation by lentiviral transduction with the WT MCM4 allele, the empty vector, or the c.70_71insG allele in P1.3 SV40 fibroblasts. Error bars indicate SEM. ***P < 0.0005, Student’s t test.