Figure 6

Mitochondrial nuclease activity. A. Fractionation PCR. A partial fragment of the mitochondrial large subunit ribosomal RNA (rRNA) or β-tubulin (BTU) was amplified by PCR, using fraction samples from wild-type and ΔAIF that contained equal amounts of DNA. N and Mt indicate nuclei/unbroken cell fraction and mitochondrial fraction, respectively. No contamination of nuclear DNA was detected in mitochondrial fraction. B. Purified mitochondria (2 μg protein) from wild-type (lane 2) and ΔAIF (lane 3) were incubated with 2 μg substrate plasmid DNA with a circular form for 30 min at 37°C in 30 μl reaction buffer containing 20 mM KCl and 50 mM MOPS (pH 6.5). Lane 4 (M) and 5 (M) indicate 100-bp ladder size marker and λHindIII-digest, respectively. The substrate DNA appears in the nicked open circular (OC), linear (L), and supercoiled (SC) forms. C. The nuclease assay was performed under various incubation times. Lane 2-4, substrate DNA was coincubated with wild-type mitochondria. Lane 5-7, substrate DNA was coincubated with ΔAIF mitochondria. Undigested sample is seen in lane 1. D. Substrate specificity of the activities. End forms of linear plasmids with 5'- or 3'-overhang or with blunt ends are indicated at the left of gel.