DNA-dependent ATPase from rat mitochondria

A DNA-dependent ATPase has been highly purified from rat liver mitochondria and characterized. The enzyme catalyzes the hydrolysis of ATP or dATP in the presence of single-stranded DNA cofactor and a divalent cation. The Km values for ATP and dATP are 0.15 mM and 0.35 mM, respectively. The enzyme activity is highly sensitive to N-ethylmaleimide. The sedimentation coefficient of the enzyme is 8.3 S in a glycerol gradient. From this and data on Sephadex G-200 gel filtration, the molecular weight of the native enzyme was calculated to be about 190,000. All the natural single-stranded DNAs tested were equally effective for the ATPase activity, but synthetic deoxyhomopolymer poly(dC) was found to be more effective than natural single-stranded DNAs. Synthetic and natural RNAs had no effect on the activity. INTRODUCTION Mitochondria have their own DNA and DNA polymerase and are able to synthesize their DNA in situ. In vertebrates, mitochondrial DNA is a closedcircular molecule of about 16 kilobase pairs. The mechanism of mitochondrial DNA synthesis has been shown to have very distinctive features. Recent studies have revealed that the mitochondrial DNA polymerase examined is identical to DNA polymerase y (1,2). However, DNA polymerase y itself cannot replicate closed-circular DNA (3), and other protein factors are expected to be involved in its replication. Although a nicking-closing enzyme has been identified in mitochondria (4), no information is available on the nature of the other protein factors involved. Recent studies on the replication systems of bacteria and phages have revealed that many enzymes or protein factors besides DNA polymerase HI holoenzyme are required for DNA replication (5). Interestingly, several of these proteins have been found to exibit DNA-dependent ATPase activity; for instance the E.coli dnaB gene product, which seems to act as a mobile promotor (6), the £. coli rep protein and DNA helicases I, II and IH, which unwind doublestranded DNA (7-9), the T7 gene 4 product, which is the primase for T7 DNA © IRL Press Limited, 1 Falconberg Court, London W1V 5FG, U.K. 1949 Nucleic Acids Research r e p l i c a t i o n (10 ) , the T4 gene 44-62 and gene 45 products, which s t imula te T4 DNA polymerase (11) , and £_. co l i DNA gyrase (12) . The involvement of DNAdependent ATPase i n prokaryot ic DNA r e p l i c a t i o n prompted us to inves t iga te an analogous p ro te in i n mi tochondr ia, because ATP has been found to be essent ia l f o r DNA synthesis i n i so la ted mitochondria (13) . In t h i s work, we p u r i f i e d a DNA-dependent ATPase from r a t mitochondria and examined the d e t a i l s o f i t s unique property of hydrolyzing ATP or dATP i n the presence of natural s ingle-st randed DNA or synthet ic poly(dC). A poss i b le r o l e of t h i s enzyme in mitochondrial DNA r e p l i c a t i o n i s discussed. EXPERIMENTAL PROCEDURES Preparat ion of mitochondria Mitochondria were prepared from the l i v e r o f adu l t r a t s as described by Fujisawa et a l . ( 14 ) , except t ha t the i s o l a t i o n medium was 0.25 M sucrose, pH 7.4 , conta in ing 1 mM Na3EDTA. L ivers were homogenized w i th 10 volumes o f the i s o l a t i o n medium i n a Potter-Elvehjem homogenizer w i th a loose ly f i t t i n g t e f l o n pest le operat ing a t less than 1,000 rpm. Nuc le i , red c e l l s , unbroken ce l l s and c e l l debr is were removed by three successive low-speed c e n t r i f u g a t ions a t 870 x g f o r 15 min, and then mitochondria were obtained from the supernatant by cen t r i f uga t i on a t 10,000 x g f o r 15 min. The mitochondr ia l p e l l e t was washed four times w i t h bu f fe r A (0.3 M sucrose, 10 mM T r i s H C l , pH 7 .4 , and 10 mM Na3EDTA} by suspension and cen t r i f uga t i on a t 10,000 x g f o r 15 min, and then stored a t -20°C u n t i l use. DNA-dependent ATPase assay The standard reac t ion mixture i n 40 p i contained 25 mM T r i s -HC l , pH 8 . 0 , 2 mM MgCl2» 1 mM d i t h i o t h r e i t o l , 5 % sucrose, 4 yg bovine serum albumin, 0.8 mM [y-P]ATP (10-15 cpm/pmole), 0.5 yg denatured c a l f thymus DNA and 5 y l of the enzyme f r a c t i o n . Incubation was ca r r ied out at 37°C fo r 60 min. The r e ac t ion was terminated by adding 100 y l each of ice-co ld 7 % perch lo r i c acid and 20 % (w/v) N o r i t . The mixture was s t i r r e d i n a Vortex mixer and c e n t r i fuged f o r 5 min a t 15,000 rpm and the r a d i o a c t i v i t y of P inorganic phosphate was measured i n a 50-yl a l i q u o t of the supernatant. The DNA-dependent ATPase a c t i v i t y was determined by measuring the d i f fe rence i n ATP hydro lys is i n the presence and absence of DNA. One u n i t o f enzyme a c t i v i t y i s def ined as the amount ca ta l yz ing the re lease o f 1 nmol o f inorganic phosphate f o r 60 min a t 37°C. Substrate [y-P]ATP was prepared by the method o f Glynn and Chappel (15) .