Substrate specificity and adenosine triphosphatase activity of the ATP-dependent deoxyribonuclease of Bacillus subtilis

Studies on the specificity of the ATP-dependent DNase of Bacillus subtilis 168, carried out with pure enzyme at the optimal conditions for its action, have shown that the substrate is double-stranded linear DNA. Linear single-stranded DNA (separated strands of B. subtilis DNA and linear phage fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked simian virus 40 and circular single-stranded fd DNAs). The double-stranded DNA can be completely hydrolysed, the limit products being, almost exclusively, mononucleotides. The presence of terminal phosphate residues in the substrate (either at the 3' or the 5' end) is not necessary for enzyme action. This DNase appears therefore to be an exonuclease processively liberating mononucleotides from both strands of the native linear DNA. ATP (indispensable for the DNase reaction) is also hydrolysed by the enzyme, to ADP and inorganic orthophosphate (Pi) in the presence of DNA. The apparent Km for ATP, in the ATPase reaction, is 0.15 mM. At high ATP concentrations, which inhibit the DNase activity, there is activation of the ATPase reaction. Three molecules of ATP are consumed for each DNA phosphodiester bond split, at optimal conditions for DNase activity.     

Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism

DNA footprinting and nuclease protection studies of PcrA helicase complexed with a 3'-tailed DNA duplex reveal a contact region that covers a significant region of the substrate both in the presence and absence of a non-hydrolysable analogue of ATP, ADPNP. However, details of the interactions of the enzyme with the duplex region are altered upon binding of nucleotide. By combining this information with that obtained from crystal structures of PcrA complexed with a similar DNA substrate, we have designed mutant proteins that are defective in helicase activity but that leave the ATPase and single-stranded DNA translocation activities intact. These mutants are all located in domains 1B and 2B, which interact with the duplex portion of the DNA substrate. Taken together with the crystal structures, these data support an 'active' mechanism for PcrA that involves two distinct ATP-dependent processes: destabilization of the duplex DNA ahead of the enzyme that is coupled to DNA translocation along the single strand product.     

Mechanism of DNA degradation by the ATP-dependent DNase from Hemophilus influenzae Rd.

The rate of production of acid-soluble material during degradation of duplex DNA by Hemophilus influenzae ATP-dependent DNAse (Hind exonuclease V) has been shown to be directly dependent upon the Mg2+ concentration in the reaction mixture. At high concentrations of Mg2+ (5 to 20 mM), DNA degradation to acid-soluble products is rapid and the rate of ATP hydrolysis is slightly depressed. At low concentrations of Mg2+ (0.1 to 0.5 mM), the enzyme rapidly hydrolyzes ATP and converts up to 35% of linear duplex DNA to single-stranded material while degrading less than 0.2% of the DNA to acid-soluble products. We refer to this enzymatic production of single-stranded DNA as the "melting" activity. Under the conditions of our assay, the initial melting reaction is processive, lasting about 70s on phage T7 DNA. Using DNAs with several different lengths, we have established that the duration of the initial reaction is dependent upon DNA length, requiring approximately 1 s per 0.18 mum. The products of the initial reaction on phage T7 DNA are somewhat heterogeneous, consisting of short duplex fragments approximately 0.5 mum long, purely single-stranded products up to 7 mum long, and longer duplex fragments 3 to 11 mum in length, some of which have single-stranded tails. Nearly half of the single-stranded material remains linked to a duplex segment of DNA after the inital processive reaction. We propose that Hind exo V initiates attack at the DNA termini and then acts in a processive manner, migrating along the DNA molecule, converting some regions to single-stranded material by the combined action of the melting activity and limited phosphodiester cleavage, while leaving other regions double-stranded. At the completion of its processive movement through a single DNA molecule, it is released and then recycles onto either intact molecules or the partially degraded products, continuing in this manner until the DNA is finally reduced to oligonucleotides.     

Purification and properties of an endonuclease from the mitochondrion of Saccharomyces cerevisiae

An endonuclease, which is found only in the mitochondrion of the yeast Saccharomyces cerevisiae, has been purified. The protein has a sedimentation coefficient of 6.3 S, equivalent to a molecular weight of 105,000. The enzyme is active at pH 7.6, when it degrades single-stranded DNA about 10-times faster than double-stranded DNA, but at pH 5.4 only double-stranded DNA is degraded. In both cases the enzyme acts endonucleolytically, breaking a single phosphodiester bond at a random location within the DNA substrate. Mn2+ or Mg2+ are required for activity; Ca2+ and Zn2+ are ineffective cofactors. Enzyme activity at pH 7.6 is severely inhibited by low concentrations of NaCl or KCl, while activity at pH 5.4 is unaffected by salt. Ethidium bromide inhibits both the DNase activity at pH 5.4 and the activity with single-stranded DNA at pH 7.6, but has no effect on the DNase activity with double-stranded DNA at pH 7.6.     

Purification and properties of adenosine 5'-triphosphate-dependent deoxyribonuclease from Thermus thermophilus HB8

An ATP-dependent DNase has been purified from Thermus thermophilus HB8 by a procedure involving streptomycin precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and heparin-agarose affinity chromatography. ATP-dependent DNase activity was separated into two distinct peaks, Peak A and Peak B, by heparin-agarose affinity chromatography. Each peak fraction was further purified by ATP-agarose affinity chromatography. Peak A and Peak B were eluted from an ATP-agarose column at 0.14 M and 0.28 M KCl, respectively, each as a single peak. Both enzyme activities require ATP and Mg2+ for the degradation of double- and single-stranded DNAs, and degrade denatured DNA about 1.5 times faster than native DNA. The two peaks are optimally active at 69 degrees C and have similar optimal pH ranges from 8.2 to 9.2. The two purified peaks were unstable on storage at -20 degrees C, but were remarkably stabilized by addition of 0.4 mg/ml bovine serum albumin. Ammonium sulfate strongly inhibits the activities of both peaks. The molecular weights of Peak A and Peak B are about 170,000 as estimated by glycerol gradient sedimentation. The average chain lengths of denatured DNA produced by Peak A and Peak B were 4.2 and 3.6, respectively, and the products were terminated by 5'-phosphoryl and 3'-hydroxyl groups. The limit-digested products of denatured DNA produced by Peak B consist of mono-, di-, tri-, tetra-, and pentanucleotides along with some larger fragments. The mode of action of both activities is processive and Peak A does not attack double-stranded circular DNA.     

Ca2+, calmodulin-dependent phosphorylation of glycogen synthase by a brain protein kinase

The inactivation of rec BC nuclease activity and simultaneously the separation of 3 DNA-dependent ATPases and an ATP-independent DNases specific for single-stranded DNA have been observed after DEAE-cellulose chromatography of cell extracts from Escherichia coli. Two of the ATPases catalyze the strand separation of duplex DNA. Reconstitution of ATP-dependent DNase activity has been carried out by the combination of the separated enzymes.     

On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli.

The deoxyribonuclease specified by the recB and recC genes of Escherichia coli (recBC DNase; exonuclease V) has been purified to near homogeneity by a new procedure. Although hydrolysis of even a single nucleotide from a duplex DNA molecule by the pure enzyme is absolutely dependent upon ATP, the extent of phosphodiester hydrolysis is strongly inhibited by ATP concentrations of 0.2 mm or greater, and the initial rate is unaffected. Under these conditions, the extent of DNA hydrolysis is proportional to enzyme concentration. In contrast, neither the rate nor the extent of hydrolysis of single-stranded DNA nor ATP is affected by high concentrations of ATP. The amount of large single-stranded polynucleotide generated by the action of the recBC DNase increases as the ATP concentration increases and, at 0.5 mM ATP, becomes equivalent to the amount of acid-soluble nucleotide formed. These findings suggest that high intracellular concentrations of ATP affect the mechanism of the recBC DNase so as to limit the extent of hydrolysis of duplex DNA, while at the same time favoring the formation of single-stranded regions within the duplex. Such regions may be essential intermediates in the recombination process.     

The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity

The DNA helicase activity associated with purified simian virus 40 (SV40) large tumor (T) antigen has been examined. A variety of DNA substrates were used to characterize this ATP-dependent activity. Linear single-stranded M13 DNA containing short duplex regions at both ends was used to show that SV40 T antigen helicase displaced the short, annealed fragment by unwinding in a 3' to 5' direction. Three different partial duplex structures consisting of 71-, 343-, and 851-nucleotide long fragments annealed to M13 single-stranded circular DNA were used to show that SV40 T antigen can readily unwind short and long duplex regions with almost equal facility. ATP and MgCl2 were required for this reaction. With the exception of GTP, dGTP, and CTP, the other common nucleoside triphosphates substituted for ATP with varied efficiency, while adenosine 5'-O-(thiotriphosphate) was inactive. The T antigen helicase activity was also examined using completely duplex DNA fragments (approximately 300 base pairs) with or without the SV40 origin sequence as substrates. In reactions containing small amounts (0.6 ng) of DNA, the ATP-dependent unwinding of duplex DNA fragments occurred with no dependence on the origin sequence. This reaction was stimulated 5- to 6-fold by the addition of the Escherichia coli single-stranded DNA-binding protein. When competitor DNA was added so that the ratio of SV40 T antigen to DNA was reduced 1000-fold, only DNA fragments containing a functional SV40 origin of replication were unwound. This reaction was dependent on ATP, MgCl2, and a DNA-binding protein, and was stimulated by inorganic phosphate or creatine phosphate. The origin sequence requirements for the unwinding reaction were the same as those for replication (the 64-base pair sequence present at T antigen binding site 2). Thus, under specified conditions, only duplex DNA fragments containing an intact SV40 core origin were unwound. In contrast, unwinding of partially duplex segments of DNA flanked by single-stranded regions can occur with no sequence specificity.     

Escherichia coli recA protein protects single-stranded DNA or gapped duplex DNA from degradation by RecBC DNase

RecA- mutants of Escherichia coli extensively degrade their DNA following UV irradiation. Most of this degradation is due to the recBC DNase, which suggests that the recA gene is involved in the control of recBC DNase in vivo. We have shown that purified recA protein inhibits the endonuclease and exonuclease activities of recBC DNase on single-stranded DNA. The extent of inhibition is dependent on the relative concentration of recA protein, recBC DNase, and the DNA substrate; inhibition is greatest when the concentrations of DNA and recBC DNase are low and the concentrations of recA protein is high. At fixed concentrations of recA protein and recBC DNase, inhibition is eliminated at high concentrations of DNA. In the presence of adenosine 5'-O-(3-thiotriphosphate), an ATP analog which stabilizes the binding of recA protein to both single- and double-stranded DNA, recA protein is a more potent inhibitor of the nuclease activities on single-stranded DNA and is a weak inhibitor of the exonuclease activity on double-stranded DNA. Inhibition of the latter is enhanced by oligodeoxynucleotides, which stimulate the binding of recA protein to double-stranded DNA. In the presence of adenosine 5'-O-(3-thiotriphosphate), recA protein also inhibits the action of exonuclease I on single-stranded DNA and of lambda exonuclease on double-stranded DNA. These observations are most consistent with the idea that recA protein protects DNA from recBC DNase by binding to DNA. RecA protein also blocks the endonucleolytic cleavage of gapped circular DNA by recBC DNase. Since both recA protein and recBC DNase have the ability under certain conditions to unwind duplex DNA and to displace strands, we looked for evidence that their combined action would enlarge gaps but found no extensive enlargement. D-loops, a putative intermediate in genetic recombination, are effectively protected against the action of recBC DNase by the E. coli single strand binding protein and by recA protein in the presence of adenosine 5'-O-(3-thiotriphosphate).     

Action of ATP-dependent DNase from Hemophilus influenzae on cross-linked DNA molecules.

The ATP-dependent DNase from Hemophilus influenzae digests double-stranded linear DNA molecules exonucleolytically while hydrolyzing large amounts of ATP to ADP. Various cross-linked linear duplex DNA molecules are partially resistant to the exonuclease action. Vaccinia DNA, containing natural terminal cross-links (probably in the form of terminal single-stranded loops), is much more slowly degraded than comparable "open-ended" DNA molecules, and ATP is consumed at a proportionately lower rate. It is postulated that the vaccinia DNA molecules undergo slow terminal cleavage by the single strand specific endonuclease activity of the enzyme, and are then rapidly degraded by the double strand exonuclease activity. Phage T7 DNA, containing an average of 100 4',5'8-trimethylpsoralen cross-links/molecule at random internal sites, is digested only to the extent of 2 to 3%. However, ATP hydrolysis continues at a linear rate long after DNA digestion has ceased. A stable enzyme-DNA complex is formed as demonstrated by co-sedimentation of DNA and ATPase activity in sucrose gradients. The hypothesis is advanced that the enzyme digests exonucleolytically to the first cross-link at each end of the DNA molecules where further movement is prevented. The enzyme then remains bound at the cross-links and functions continuously as an ATPase.     

Purification and properties of the deoxyribonucleic acid polymerase induced by vaccinia virus.

The vaccinia virus-induced DNA polymerase has been purified about 500-fold from a cytoplasmic extract of vaccinia-infected HeLa cells. Analysis of the purified fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide of 110,000 daltons, which is greater than 95% pure. This polypeptide co-sediments with polymerase activity through a glycerol gradient. The sedimentation coefficient of the enzyme is 6.3 S, and its Stokes radius is 4.6 nm. The molecular weight of the native enzyme derived from these values is 115,000. Vaccinia polymerase is therefore a single large polypeptide of 110,000 to 115,000 daltons. The purified fraction has no significant endonuclease activity, but a strong exonuclease activity co-purifies with polymerase activity through every step in the isolation. The polymerase and exonuclease activities are inactivated at 45 degrees C at the same rate. It is likely, therefore, that both activities are catalyzed by the same polypeptide. The exonuclease hydrolyzes DNA predominantly in the 3' leads to 5' direction, to produce 5' mononucleotides. The exonuclease degrades single-stranded DNA more rapidly than duplex DNA, and the rate of digestion of both single-stranded and double-stranded DNA increases as the size of the substrate decreases. Single-stranded circular DNA is a potent inhibitor of the exonuclease activity, but duplex circular DNA has no significant effect on its activity.     

DNA helicase and nucleoside-5'-triphosphatase activities of polyoma virus large tumor antigen

Polyoma virus large tumor antigen (PyV T antigen) has been purified to near homogeneity by immunoaffinity column chromatography. We have detected DNA helicase and ATPase (nucleoside-5'-triphosphatase) activities in the purified PyV T antigen fraction and characterized these activities. The ATPase activity was stimulated about 2-fold by poly(dT), which was the most effective stimulator among the synthetic polynucleotides tested. Natural nucleic acids, such as calf thymus native and heat-denatured DNA, and single-stranded circular fd DNA were also effective, but the degree of stimulation was less than 1.5-fold. The basal and poly(dT)-stimulated ATPase activities showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optima. The preference for nucleoside 5'-triphosphates was ATP, dATP greater than CTP, UTP much greater than GTP. The only difference observed between the two activities was salt sensitivity. The basal ATPase activity was resistant to KC1 up to 300 mM. In contrast, poly-(dT)-stimulated activity was reduced to the level of basal activity at 300 mM KC1. DNA helicase activity required divalent cations and was dependent on hydrolysis of ATP. The activity showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optimum as the two ATPase activities, and the salt sensitivity of DNA helicase activity was similar to that of poly(dT)-stimulated ATPase activity. The helicase activity was inhibited competitively by the addition of single-stranded or double-stranded DNA, and a relatively high inhibitory activity was observed with poly [d(A-T)]. The PyV T antigen helicase was found to migrate in the 3' to 5' direction along the DNA strand to which the protein bound.     

Properties of Bacillus subtilis ATP-dependent deoxyribonuclease.

A purification procedure described previously resulting in electrophoretically pure Bacillus subtilis ATP-dependent DNAse has now been modified by adding a fractionation stage with Polymin P to permit large-scale isolation of the enzyme. It has been found that the enzyme molecule (Mr = 300000) consists of two large subunits with Mr 155000 and 140000. The purified enzyme has three activities: (1) DNAse on linear single-stranded and double-stranded DNAs (2) DNA-unwinding and (3) ATPase. Circular DNAs were not affected by the enzyme. Study of the dependence of these activities on temperature, pH, and ATP and Mg2+ concentrations has revealed two different states of the enzyme. At low ATP concentrations and alkaline pH, it showed chiefly nuclease action, degrading considerable amounts of DNA to small fragments five residues long on average. At higher ATP concentrations and neutral pH (more physiological conditions) it predominantly unwound DNA. Simultaneously it cut preferentially one of the duplex strands to fragments more than 1000 residues in length. The results obtained suggest that the energy of the enzyme-cleaved ATP is mainly expended on unwinding rather than on degrading DNA molecules.     

Translocation of Escherichia coli recA protein from a single-stranded tail to contiguous duplex DNA

Duplex DNA with a contiguous single-stranded tail was nearly as effective as single-stranded DNA in acting as a cofactor for the ATPase activity of recA protein at neutral pH and concentrations of MgCl2 that support homologous pairing. The ATP hydrolysis reached a steady state rate that was proportional to the length of the duplex DNA attached to a short 5' single-stranded tail after a lag. Separation of the single-stranded tail from most of the duplex portion of the molecule by restriction enzyme cleavage led to a gradual decline in ATP hydrolysis. Measurement of the rate of hydrolysis as a function of DNA concentration for both tailed duplex DNA and single-stranded DNA cofactors indicated that the binding site size of recA protein on a duplex DNA lattice, about 4 base pairs, is similar to that on a single-stranded DNA lattice, about four nucleotides. The length of the lag phase preceding steady state hydrolysis depended on the DNA concentration, length of the duplex region, and the polarity of the single-stranded tail, but was comparatively independent of tail length for tails over 70 nucleotides in length. The lag was 5-10 times longer for 3' than for 5' single-stranded tailed duplex DNA molecules, whereas the steady state rates of hydrolysis were lower. These observations show that, after nucleation of a recA protein complex on the single-stranded tail, the protein samples the entire duplex region via an interaction that is labile and not strongly polarized.     

Isolation and comparison of two molecular species of the BAL 31 nuclease from Alteromonas espejiana with distinct kinetic properties

The extracellular nuclease from Alteromonas espejiana sp. BAL 31 can be isolated as two distinct proteins, the "fast" (F) and "slow" (S) species, both of which have been purified to homogeneity. The F and S species of the nuclease have molecular weights, respectively, of 109 X 10(3) and 85 X 10(3), and both are single polypeptide chains with an isoelectric pH near 4.2. Both species catalyze the degradation of single-stranded and linear duplex DNAs to 5'-mononucleotides. The degradation of linear duplex DNA occurs through a terminally directed hydrolysis mechanism that results in the removal of nucleotides from both the 3' and 5' ends. Apparent Michaelis constants (Km) have been obtained for the exonuclease activities of both species and for the activity against single-stranded DNA of the S species. The Km for the hydrolysis of single-stranded DNA catalyzed by the F species has not been obtained because the reaction velocity was maximal even at the lowest substrate concentrations accessible in the photometric assay. The ratio of the turnover numbers for the exonuclease activities of the two species indicates that the F species will shorten linear duplex DNA at a rate 27 +/- 5 (S.D.) times faster than an equimolar concentration of the S species in the limit of high substrate concentration, while the corresponding ratio for the activities against single-stranded DNA (1.2 +/- 0.1) shows that the two species are similar with respect to hydrolysis of this substrate. In the limit of high substrate concentrations, the F and S species break phosphodiester bonds in single-stranded DNA at rates 1.3 +/- 0.3 and 33 +/- 2 times those for the exonucleolytic degradation of linear duplex DNA, respectively. It has not been established whether the two species are physically related.     

A DNA helicase from human cells.

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.     

The distribution of Escherichia coli recA protein bound to duplex DNA with single-stranded ends

A short single-stranded tail on one end of an otherwise duplex DNA molecule enables recA protein, in the presence of ATP and MgCl2, to form a complex with the DNA which extends into the duplex portion of the molecule. Nuclease protection studies at a concentration of MgCl2 which permits homologous pairing showed that cleavage by restriction endonucleases at sites throughout the duplex region was inhibited, whereas digestion by DNase I was not affected. These results indicate that recA protein binds to the duplex portion of tailed DNA allowing access by DNase I to a random sample of the many sites at which it cleaves, but providing limited protection of the relatively rare restriction sites. Electron microscopy revealed that the recA nucleoprotein complex with duplex DNA is indeed a segmented or interrupted filament that, with time, extends further from the single-stranded tail into the duplex region. recA protein binding extended into the duplex region more rapidly for duplexes with 5' tails than for those with 3' tails. These observations show that recA protein translocates from a single-stranded region into duplex DNA in the form of a segmented filament by a mechanism that is not strongly polarized.     

Deoxyribonuclease II purified from Euglena gracilis SM-ZK, a chloroplast-lacking mutant: comparison with porcine spleen deoxyribonuclease II

An acid deoxyribonuclease was extracted from Euglena gracilis SM-ZK, a chloroplast-lacking strain, by homogenizing the cells in 50 mM sodium acetate (pH 4.6). The enzyme was then purified by heat treatment and a series of chromatographic separations. The molecular mass of the Euglena acid DNase was estimated to be 45 kDa by sensitive activity staining in an SDS-polyacrylamide gel using SYBR Green. Treatment of the Euglena enzyme with a reducing agent prior to electrophoresis destroyed its DNase activity in the gel, indicating that disulfide bridging is essential for its enzyme activity. Nucleolytic properties of this enzyme are essentially the same as to those of porcine DNase II. The Euglena enzyme acts on both double-stranded (ds) and single-stranded DNA, but acts preferentially on dsDNA with an optimum pH at approximately 5.3. EDTA did not inhibit its enzyme activity. Euglena DNase makes double-strand breaks in circular DNA substrate and generates a terminus with 3'-phosphate and 5'-OH. These results indicate that the Euglena acid DNase is in fact a member of the DNase II family.     

Exonuclease activity from Pseudomonas aeruginosa which is missing in phenotypically restrictionless mutants.

A phenotypically restrictionless strain of Pseudomonas aeruginosa was found to lack a deoxyribonuclease specific for linear duplex DNA. The purified enzyme had an optimum pH of 8.5, required MgCl2 (10 mM) for maximum activity, and did not require ATP. Neither the degradation of heat-denatured DNA nor the degradation of bacteriophage F116 DNA was detected. The genome of bacteriophage F116 was shown to possess single-stranded terminal regions, which account for the resistance to degradation and for the ability of the phage to transfect restriction-proficient strains.     

Separation of ATP-dependent DNAse to ATPase and DNAse

A 250-fold purified ATP-dependent DNase from Bacillus cereus has been separated to DNA-dependent ATPase I and II and a DNase specific for single-stranded DNA (ssDNase) by means of high resolution of DEAE cellulose chromatography. Simultaneously with the separation of ATPase and ssDNase, a decrease in ATP-dependent DNase activity was observed. Complete separation resulted in the total loss of ATP-dependent DNase activity. Reconstitution of ATP-stimulated DNase activity was dependent on the ratio of the combined ATPase II and ssDNase.