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.     

Biochemical characterization of an exonuclease from Arabidopsis thaliana reveals similarities to the DNA exonuclease of the human Werner syndrome protein

The human Werner syndrome protein (hWRN-p) possessing DNA helicase and exonuclease activities is essential for genome stability. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis genome contains a small open reading frame (ORF) (AtWRNexo) with homology to the exonuclease domain of hWRN-p. Expression of this ORF in Escherichia coli revealed an exonuclease activity for AtWRN-exo-p with similarities but also some significant differences to hWRN-p. The protein digests recessed strands of DNA duplexes in the 3' --> 5' direction but hardly single-stranded DNA or blunt-ended duplexes. In contrast to the Werner exonuclease, AtWRNexo-p is also able to digest 3'-protruding strands. DNA with recessed 3'-PO4 and 3'-OH termini is degraded to a similar extent. AtWRNexo-p hydrolyzes the 3'-recessed strand termini of duplexes containing mismatched bases. AtWRNexo-p needs the divalent cation Mg2+ for activity, which can be replaced by Mn2+. Apurinic sites, cholesterol adducts, and oxidative DNA damage (such as 8-oxoadenine and 8-oxoguanine) inhibit or block the enzyme. Other DNA modifications, including uracil, hypoxanthine and ethenoadenine, did not inhibit AtWRNexo-p. A mutation of a conserved residue within the exonuclease domain (E135A) completely abolished the exonucleolytic activity. Our results indicate that a type of WRN-like exonuclease activity seems to be a common feature of the DNA metabolism of animals and plants.     

T7 gene 6 exonuclease has an RNase H activity.

T7 gene 6 exonuclease has been shown to have an RNase H activity as well as a double-strand specific DNase activity by the following experiments: The RNase H activity coelutes with the DNase activity from DEAE-cellulose, phosphocellulose, hydroxyapatite, and Sephadex G-200 columns. Gene 6 exonuclease specified by a T7 strain with a temperature sensitive mutation in gene 6 has an extremely heat-labile RNase H activity as well as a heat-labile DNase activity. T7 gene 6 exonuclease degrades the RNA region of a poly(A) . poly(dT) hybrid polymer exonucleolytically from the 5' terminus, releasing a ribonucleoside 5'-monophosphate product. When the RNA strand of a 0X174 RNA . DNA hybrid molecule synthesized with E. coli RNA polymerase is degraded, a ribonucleoside triphosphate is produced from the 5'-triphosphate terminus. Participation of T7 gene 6 exonuclease in the removal of primer RNA in discontinuous replication of T7 DNA is discussed.     

5' to 3' single strand DNA exonuclease activity in a preparation of human Ku protein

We describe a novel 5' to 3' single-strand exonuclease activity exhibited by a Ku preparation purified from a human cell line. The enzyme removes 5' single-strand extensions from duplex DNA molecules. The exonuclease and helicase activities respond reciprocally to changes in ATP concentrations: Nuclease activity is inhibited at the ATP concentrations that are optimal for the helicase. The exonuclease activity does not require divalent cations. The potential implications of the exonuclease activity findings for repair of double-strand breaks and recombination processes are discussed.     

Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae

An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.     

Purification and characterization of Rad3 ATPase/DNA helicase from Saccharomyces cerevisiae

The Rad3 ATPase/DNA helicase was purified to physical homogeneity from extracts of yeast cells containing overexpressed Rad3 protein. The DNA helicase can unwind duplex regions as short as 11 base pairs in a partially duplex circular DNA substrate and does so by a strictly processive mechanism. On partially duplex linear substrates, the enzyme has a strict 5'----3' polarity with respect to the single strand to which it binds. Nicked circular DNA is not utilized as a substrate, and the enzyme requires single-stranded gaps between 5 and 21 nucleotides long to unwind oligonucleotide fragments from partially duplex linear molecules. The enzyme also requires duplex regions at least 11 base pairs long when these are present at the ends of linear molecules. Rad3 DNA helicase activity is inhibited by the presence of ultraviolet-induced photoproducts in duplex regions of partially duplex circular molecules.     

Single strand annealing and ATP-independent strand exchange activities of yeast and human DNA2: possible role in Okazaki fragment maturation

The Dna2 protein is a multifunctional enzyme with 5'-3' DNA helicase, DNA-dependent ATPase, 3' exo/endonuclease, and 5' exo/endonuclease. The enzyme is highly specific for structures containing single-stranded flaps adjacent to duplex regions. We report here two novel activities of both the yeast and human Dna2 helicase/nuclease protein: single strand annealing and ATP-independent strand exchange on short duplexes. These activities are independent of ATPase/helicase and nuclease activities in that mutations eliminating either nuclease or ATPase/helicase do not inhibit strand annealing or strand exchange. ATP inhibits strand exchange. A model rationalizing the multiple catalytic functions of Dna2 and leading to its coordination with other enzymes in processing single-stranded flaps during DNA replication and repair is presented.     

DNA strand transfer catalyzed by the 5'-3' exonuclease domain of Escherichia coli DNA polymerase I.

A protein which promotes DNA strand transfer between linear double-stranded M13mp19 DNA and single-stranded viral M13mp19 DNA has been isolated from recA- E.coli. The protein is DNA polymerase I. Strand transfer activity residues in the small fragment encoding the 5'-3' exonuclease and can be detected using a recombinant protein comprising the first 324 amino acids encoded by polA. Either the recombinant 5'-3' exonuclease or intact DNA polymerase I can catalyze joint molecule formation, in reactions requiring only Mg2+ and homologous DNA substrates. Both kinds of reactions are unaffected by added ATP. Electron microscopy shows that the joint molecules formed in these reactions bear displaced single strands and therefore this reaction is not simply promoted by annealing of exonuclease-gapped molecules. The pairing reaction is also polar and displaces the 5'-end of the non-complementary strand, extending the heteroduplex joint in a 5'-3' direction relative to the displaced strand. Thus strand transfer occurs with the same polarity as nick translation. These results show that E.coli, like many eukaryotes, possesses a protein which can promote ATP-independent strand-transfer reactions and raises questions concerning the possible biological role of this function.     

Probing the relationship between RNA-stimulated ATPase and helicase activities of HCV NS3 using 2'-O-methyl RNA substrates

The hepatitis C virus (HCV) NS3 protein contains an amino terminal protease (NS3 aa. 1-180) and a carboxyl terminal RNA helicase (NS3 aa. 181-631). NS3 functions as a heterodimer of NS3 and NS4A (NS3/4A). NS3 helicase, a nucleic acid stimulated ATPase, can unwind RNA, DNA, and RNA:DNA duplexes, provided that at least one strand of the duplex contains a single-stranded 3' overhang (this strand of the duplex is referred to as the 3' strand). We have used 2'-O-methyl RNA (MeRNA) substrates to study the mechanism of NS3 helicase activity and to probe the relationship between its helicase and RNA-stimulated ATPase activities. NS3/4A did not unwind double-stranded (ds) MeRNA. NS3/4A unwinds hybrid RNA:MeRNA duplex containing MeRNA as the 5' strand but not hybrid duplex containing MeRNA as the 3' strand. The helicase activity of NS3/4A was 50% inhibited by 40 nM single-stranded (ss) RNA but only 35% inhibited by 320 nM ss MeRNA. Double-stranded RNA was 17 times as effective as double-stranded MeRNA in inhibiting NS3/4A helicase activity, while the apparent affinity of NS3/4A for ds MeRNA differed from ds RNA by only 2.4-fold. However ss MeRNA stimulated NS3/4A ATPase activity similar to ss RNA. These results indicate that the helicase mechanism involves 3' to 5' procession of the NS3 helicase along the 3' strand and only weak association of the enzyme with the displaced 5' strand. Further, our findings show that maximum stimulation of NS3 ATPase activity by ss nucleic acid is not directly related to procession of the helicase along the 3' strand.     

Electron microscopic analysis of DNA forks generated by Escherichia coli DNA helicase II

T7 phage DNA eroded with lambda exonuclease (to create 3'-protruding strands) or exonuclease III (to create 5'-protruding strands) was treated under unwinding assay conditions with DNA helicase II. Single-stranded DNA-binding protein (of Escherichia coli or phage T4) was added to disentangle the denatured DNA and the complexes were examined in the electron microscope. DNA helicase II complexes filtered through a gel column before assay retain the ability to generate forks suggesting that DNA helicase II unwinds in a preformed complex by translocating along the bound DNA strand. The enzyme initiates preferentially at the ends of the lambda-exonuclease-treated duplexes and is found at a fork on the initially protruding strand. It also initiates at the ends of the exonuclease-III-treated duplexes where, as with approximately 5% of the forks traceable back to a single-stranded gap, it is found on the initially recessed strand. The results are consistent with the view that DNA helicase II unwinds in the 3'-5' direction relative to the bound strand. They also confirm that the enzyme can initiate at the end of a fully base-paired strand. At a fork, DNA helicase II is bound as a tract of molecules of approximately 110 nm in length. Tracts of enzyme assemble from non-cooperatively bound molecules in the presence of ATP. During unwinding, DNA helicase II apparently can translocate to the displaced strand which conceivably can deplete the leading strand of the enzyme. Continued adsorption of enzyme to DNA might replenish forks arrested by strand switch of the unwinding enzyme.     

Biochemical and physical characterization of exonuclease V from Escherichia coli. Comparison of the catalytic activities of the RecBC and RecBCD enzymes

Biochemical evidence is presented that confirms exonuclease V of Escherichia coli consists of three distinct subunits encoded by the recB, recC, and recD genes. The recD gene encodes a Mr 60,000 polypeptide and physically maps 3' to the recB structural gene. The role of the recD subunit in exonuclease V function has been examined by comparing the catalytic activities of the purified RecBCD enzyme with the RecBC enzyme. The RecBC enzyme retains significant levels of DNA-dependent ATPase activity and DNA helicase activity. Endonucleolytic activity on single-stranded covalently closed DNA becomes ATP-dependent. Exonucleolytic activity on either single- and double-stranded DNA was not detected. Taken together with the phenotypic properties of recD null mutants, it appears that the exonucleolytic activities of the RecBCD enzyme are not required for genetic recombination and the repair of either UV-induced photoproducts or mitomycin C-generated DNA cross-links, but are essential for the repair of methyl methanesulfonate-induced methylation.     

Bacteriophage P22 Abc2 protein binds to RecC increases the 5' strand nicking activity of RecBCD and together with lambda bet, promotes Chi-independent recombination

Bacteriophage P22 Abc2 protein binds to the RecBCD enzyme from Escherichia coli to promote phage growth and recombination. Overproduction of the RecC subunit in vivo, but not RecB or RecD, interfered with Abc2-induced UV sensitization, revealing that RecC is the target for Abc2 in vivo. UV-induced ATP crosslinking experiments revealed that Abc2 protein does not interfere with the binding of ATP to either the RecB or RecD subunits in the absence of DNA, though it partially inhibits RecBCD ATPase activity. Productive growth of phage P22 in wild-type Salmonella typhimurium correlates with the presence of Abc2, but is independent of the absolute level of ATP-dependent nuclease activity, suggesting a qualitative change in the nature of Abc2-modified RecBCD nuclease activity relative to the native enzyme. In lambda phage crosses, Abc2-modified RecBCD could substitute for lambda exonuclease in Red-promoted recombination; lambda Gam could not. In exonuclease assays designed to examine the polarity of digestion, Abc2 protein qualitatively changes the nature of RecBCD double-stranded DNA exonuclease by increasing the rate of digestion of the 5' strand. In this respect, Abc2-modified RecBCD resembles a RecBCD molecule that has encountered the recombination hotspot Chi. However, unlike Chi-modified RecBCD, Abc2-modified RecBCD still possesses 3' exonuclease activity. These results are discussed in terms of a model in which Abc2 converts the RecBCD exonuclease for use in the P22 phage recombination pathway. This mechanism of P22-mediated recombination distinguishes it from phage lambda recombination, in which the phage recombination system (Red) and its anti-RecBCD function (Gam) work independently.     

Mechanism of replication of bacteriophage phi X174 XX. Sensitivity of nascent DNA to single-strand-specific nucleases.

We reported earlier that dephosphorylated nascent phi X174 viral strand DNA molecules were less extensively degraded from the 5' end by spleen exonuclease than were non-nascent molecules. Experiments described here revealed that the insensitivity to the 5'-OH end-specific nuclease was more evident among the longer molecules in the population than among the shorter, all of the molecules being less than unit length in size. The smallest molecules in the population were about as sensitive to the enzyme as the control molecules and hence must possess unblocked 5'-terminal nucleotides. Degradation of the nascent DNA with the 3' end-specific snake venom phosphodiesterase revealed only a small enrichment for [3H]thymidine near the 3' end, seemingly insufficient to account completely for the apparent insensitivity of the longer molecules to spleen exonuclease. When the nascent molecules were isolated without the use of proteolytic enzymes, some pronase-sensitive material was found associated with the DNA, particularly the longer molecules. We suggest that the resistance of the longer nascent (pronase-treated) molecules to spleen exonuclease occurs because they have remnants of the viral gene A or A* protein covalently bound to the 5' end.     

Adenovirus terminal protein protects single stranded DNA from digestion by a cellular exonuclease.

Adenovirus 5 DNA-protein complex is isolated from virions as a duplex DNA molecule covalently attached by the 5' termini of each strand to virion protein of unknown function. The DNA-protein complex can be digested with E. coli exonuclease III to generate molecules analogous to DNA replication intermediates in that they contain long single stranded regions ending in 5' termini bound to terminal protein. The infectivity of pronase digested Adenovirus 5 DNA is greatly diminished by exonuclease III digestion. However, the infectivity of the DNA-protein complex is not significantly altered when up to at least 2400 nucleotides are removed from the 3' ends of each strand. This indicates that the terminal protein protects 5' terminated single stranded regions from digestion by a cellular exonuclease. DNA-protein complex prepared from a host range mutant with a mutation mapping in the left 4% of the genome was digested with exonuclease III, hybridized to a wild type restriction fragment comprising the left 8% of the genome, and transfected into HeLa cells. Virus with wild type phenotype was recovered at high frequency.     

Mechanism of 3'' to 5'' exonuclease associated with phage T5-induced DNA polymerase: processiveness and template specificity.

T5-induced DNA polymerase has an associated 3' to 5' exonuclease activity. Both single-stranded and duplex DNA are hydrolyzed by this enzyme in a quasi-processive manner. This is indicated by the results of polymer-challenge experiments utilizing product analysis techniques. Due to the quasi-processive mode of hydrolysis, the kinetics of label release from the 3'-terminally labeled oligonucleotide substrates, annealed to complementary homopolymers, show an initial high rate of hydrolysis. In the case of both single-stranded and duplex DNA substrates, hydrolysis seems to continue, at best, up to the point where the enzyme is five or six nucleotides away from the 5-end. The enzyme carries out mismatch repair, as evidenced by experiments with primer molecules containing improper base residues at the 3'-OH terminus. Control experiments with complementary base residues at the 3'-end indicate that extensive removal of terminal residue takes place in the presence of dNTP's only when such residues are "improper" in the Watson-Crick sense.     

Werner syndrome exonuclease catalyzes structure-dependent degradation of DNA

Werner syndrome (WS) is an autosomal recessive disease characterized by early onset of many features of aging, by an unusual spectrum of cancers, and by genomic instability. The WS protein (WRN) possesses 3′→5′ DNA helicase and associated ATPase activities, as well as 3′→5′ DNA exonuclease activity. Currently, WRN is the only member of the widely distributed RecQ DNA helicase family with documented exonuclease activity. It is not known whether deficiency of the exonuclease or helicase/ATPase activities of WRN, or all of them, is responsible for various elements of the WS phenotype. WRN exonuclease has limited homology to Escherichia coli RNaseD, a tRNA processing enzyme. We show here that WRN preferentially degrades synthetic DNA substrates containing alternate secondary structures, with an exonucleolytic mode of action suggestive of RNaseD. We present evidence that structure-dependent binding of WRN to DNA requires ATP binding, while DNA degradation requires ATP hydrolysis. Apparently, the exonuclease and ATPase act in concert to catalyze structure-dependent DNA degradation. We propose that WRN protein functions as a DNA processing enzyme in resolving aberrant DNA structures via both exonuclease and helicase activities.     

Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks

gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed.     

Both strands of polyoma DNA are replicated discontinuously with ribonucleotide primers in vivo

Nascent polyoma DNA molecules were isolated after pulse-labeling of infected murine 3T6 cells with [3H]thymidine. The extent of digestion of these DNA molecules by spleen exonuclease was increased by exposure to alkali or RNase, suggesting that ribonucleotides were present at or near the 5' terminal of the newly synthesized pieces of DNA. Intermediates shorter than 300 nucleotides were hybridized to the separated strands of restriction enzyme fragments of the polyoma genome: 2.5 to 3-fold more radioactivity was found in the strand whose synthesis is necessarily discontinuous (the lagging strand) than in the strand whose synthesis is potentially continuous (the leading strand) than in the strand whose synthesis is potentially continuous (the leading strand). Separation of the strands of [5'-32P]DNA molecules showed that the excess [3H]thymidine in lagging-strand molecules was not simply the result of an increased number of molecules. Therefore, assuming equivalent efficiencies of labeling, lagging-strand pieces must be slightly longer than those with leading-strand polarity. The presence of ribonucleotides on the 5' termini of molecules with both leading- and lagging-strand polarity was demonstrated by (i) release of 32P-ribonucleoside diphosphates upon alkaline hydrolysis of [5'-32P]DNA separated according to replication polarity and (ii) the change in the degree of self-annealing of nascent molecules upon preferential degradation of DNA molecules possessing initiator RNA moieties by spleen exonuclease. We conclude that replication of polyoma DNA in vivo occurs discontinuously on both sides of the growing fork, using RNA as the major priming mechanism.     

Structural and enzymological characterization of a deoxyribonucleic acid dependent adenosine triphosphatase from KB cell nuclei

We have purified to near homogeneity the single DNA-dependent ATPase activity that we have identified in extracts of KB cell nuclei. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75000 daltons that was coincident with the profile of ATPase activity resolved by the final step of agarose-ATP chromatography or by isoelectric focusing. The enzyme has a pI of 8.5, a Stokes' radius by gel filtration of 3.8 nm, and a sedimentation coefficient in high salt of 5.3 S. At low ionic strength the enzyme activity sediments at 7.0 S, suggesting that it may dimerize under these conditions. The purified enzyme has a specific activity of 5.9 X 10(5) nmol of ATP hydrolyzed per h per mg of protein and is devoid of endonuclease, exonuclease, RNA or DNA polymerase, nicking-closing, and gyrase activities at exclusion limits of 10(-6)-10(-8) of the ATPase activity. The enzyme can hydrolyze only ATP or dATP, to generate ADP or dADP plus Pi, but the other NTPs and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ greater than Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Single-stranded DNA or deoxyhomopolymers are most effective, but blunt-ended linear and nicked circular duplex DNA molecules are also used at Vmax values approximately 20% of that obtained with single-stranded DNA. Intact duplex DNA and polyribonucleotides are unable to support ATP hydrolysis. Velocity gradient sedimentation studies corroborate the interpretations of the kinetic analyses and demonstrate enzyme binding to single-stranded DNA and nicked duplex DNA but not to intact duplex DNA. Although we have not succeeded directly in demonstrating DNA unwinding by this protein, preliminary results suggest that in the presence of ATP, the ATPase can stimulate the reactivity of homogeneous human DNA polymerases alpha and beta on nicked duplex DNA substrates.