FIGURE 1:

Comparison of efficiency of classical NHEJ in cell-free extracts and in mitochondrial extracts prepared from various rat tissues. (A, B) Evaluation of purity of mitochondrial extracts. Mitochondrial and cell-free extracts were prepared from rat tissues (brain, testes, spleen, kidney) and analyzed for specific markers by Western blotting. CE, cytoplasmic extract; ME, mitochondrial extract. CEs and MEs were probed with VDAC and cytochrome C (mitochondrial markers), with β-actin as loading control (A). PCNA and β-actin (nuclear and CFE markers, respectively) were used as the markers to test the purity of fractions. (C) Schematic representation of the oligomeric substrates (5′ compatible, noncompatible [5′-5 and 5′-3′], and blunt ends) mimicking endogenous DSBs used for the study. (D–G) Bar diagrams showing quantification of the end-joined products from mitochondrial extracts from the brain (D), testes (E), spleen (F), and kidney (G) using 5′-compatible, noncompatible, and blunt ends. WCE extracts from respective rat tissues were used as positive control. For NHEJ assay, 5-μg extracts were incubated with [γ-³²P]ATP-labeled oligomeric DNA substrate for 2 h at 30°C (testis) or 37°C. The reaction products were deproteinized and resolved on 8% denaturing PAGE. For quantification, the highest photostimulated light unit (PSLU) value of end joining was taken as 100%, and the relative efficiency between different extracts was calculated. Experiments were repeated a minimum of three times.