DNA biosynthesis in mitochondria: partial purification of a distinct DNA polymerase from isolated rat liver mitochondria.

Abstract

It is becoming increasingly evident that mitochondria possess a considerable degree of autonomy in their process of biogenesis.' That mitochondria canl also synthesize their ownl DNA has been suggested by in vivo autoradiographic2-6 and biochemical studies,7'9 and has been proved conielusively by biochemical studies on isolated mitochondria.10-12 In this report we now presenit evidence that the mitochondrion- contains its own. I)NA polymerase, distinct from that in the nucleus. Experimental.--Preparation of mitochondrial DNA polymerase: Rat liver mitochondria were isolated from 175-200-gmn rats according to the method of Schneider and Hogeboom3 as described previously. A typical preparation (100 gm of liver) yielded about 1 gm of mitochondrial protein. The packed pellet was frozen in a dry ice-acetone bath and ground with levigated alumina (2 parts alumina to I part wet weight of mitochondria). All further operations were carried out at 0-4?. The paste was extracted with 20-25 ml of 0.05 ill Tris buffer, pH 8.0 (25?) containing 0.02 ll' Mg acetate, 0.005 Ml ,B-mercaptoethanol, 0.001 ll/ EDTA, and 1._ JIL NaCl, and was centrifuged 60 min at 165,000 X g (Spinco 50 Ti rotor). The supernatant fluid was passed through a Sephadex G-25 column (3 X 20 cm) equilibrated with Tris-MVIE (0.025 11J Tris pH 8.0 [25?I and 0.005 l1 ,-mercaptoethanol). The desalted extract (fraction \t-I) was stored at -125?C. Fraction Mt-I was thawed and slowNly brought to 25%0 saturation with satuirated (NH4)2SO4 adjusted to pH 7.0 with NH13. After centrifugation, the supernatant solution was brought to 43%0 saturation. The precipitate was collected, dissolved in Tris-M\E buffer, and dialyzed in a rapid dialyzer14 against 3-4 changes of the Tris-M\IE for 3-4 hr (fractiolln A\It-Il). A DEAE-cellulose column (0.9 X 7 cm) was washed, and fraction Mt-II (7-20 lug of protein per run) was applied anid fraction collecting (2.4 ml) begun immediately. The column was then washed with about 35 ml of Tris-ME and eluted with 200 ml of a linear NaCl gradient (0-0.35 Jj/f NaCl in Tris-M?\IE) at a flow rate of about 0.8 ml/min. The active fractions were pooled and concentrated by precipitation at 50%0 saturated (NH4)2S04. The dialyzed fraction (Mt-11) was stored at 40 and was used for periods up to 2 weeks. Preparation of nuclear DNAA polymerase: Rat liver nuclei were prepared by the method of Blobel and Potter15 nmodified as follows: (1) their TMK buffer was changed to 0.05 Ml Tris pH 8.0 (25?), 0.005 I'I Mg acetate, 0.025 lI[ KCl, 0.005 Al.- 0-mercaptoethanol, and 0.001 AlV EDTA; (2) the larger-volume Spinlco SW25.2 lrotor replaced the SW39 and the centrifugation time was increased to I hr at 25,000 rpm; (3) the sucrose was then decanted and the upper 75% of the tube was cut off. The nuclear pellets were combined and resuspended in 75 ml TM1K. A typical preparation (180 gm of liver) runm in 6 batches yielded about 850 mg of protein. The nuclear suspension was sonicated 1-2 min until all nuclei were lysed. NaCl (4 ll) was slowly added to a concentration of 1 Ml/f. The extract was dialyzed 4-6 hr against three changes of TMK containing 0.15 211 NaCl. (The complexes which occur betweeni I)NA and polymerase or histone are dissociated by 1.0 M/f NaCl. Subsequent reduction of the NaCl concentrationi to 0.15 M results in the precipitation of DNA-histone complex while avoiding reassociation of the DNA with the polymerase.) The precipitate was removed by centrifugation and the supernatant solution constitutes fraction Nc-I.