Escherichia coli Rep protein and helicase IV. Distributive single-stranded DNA-dependent ATPases that catalyze a limited unwinding reaction in vitro.

Rep protein and helicase IV, two DNA-dependent adenosine 5'-triphosphatases with helicase activity, have been purified from Escherichia coli and characterized. Both enzymes exhibit a distributive interaction with single-stranded DNA as DNA-dependent ATPases in a reaction that is relatively resistant to increasing NaCl concentration and sensitive to the addition of E. coli single-stranded DNA binding protein (SSB). The helicase reaction catalyzed by each protein has been characterized using a direct unwinding assay and partial duplex DNA substrates. Both Rep protein and helicase IV catalyzed the unwinding of a duplex region 71 bp in length. However, unwinding of a 119-bp or 343-bp duplex region was substantially reduced compared to unwinding of the 71-bp substrate. At each concentration of protein examined, the number of base pairs unwound was greatest using the 71-bp substrate, intermediate with the 119-bp substrate and lowest using the 343-bp substrate. The addition of E. coli SSB did not increase the fraction of the 343-nucleotide fragment unwound by Rep protein. However, the addition of SSB did stimulate the unwinding reaction catalyzed by helicase IV approximately twofold. In addition, ionic strength conditions which stabilize duplex DNA (i.e. addition of MgCl2 or NaCl), markedly inhibited the helicase reaction catalyzed by either Rep protein or helicase IV while having little effect on the ATPase reaction. Thus, these two enzymes appear to share a common biochemical mechanism for unwinding duplex DNA which can be described as limited unwinding of duplex DNA. Taken together these data suggest that, in vitro, and in the absence of additional proteins, neither Rep protein nor helicase IV catalyzes a processive unwinding reaction.     

Identification and purification of a protein that stimulates the helicase activity of the Escherichia coli Rep protein

A polypeptide (Mr = 15,000) has been purified from Escherichia coli cell extracts that significantly stimulates the duplex DNA unwinding reaction catalyzed by E. coli Rep protein. The Rep helicase unwinding reaction was stimulated by as much as 20-fold, upon addition of the stimulatory protein, using either a 71-base pair or a 343-base pair partial duplex DNA molecule as a substrate. The purified Rep helicase stimulatory protein (RHSP) had no intrinsic helicase activity or ATP hydrolysis activity and did not stimulate the single-stranded DNA-dependent ATP hydrolysis reaction catalyzed by Rep protein. It is likely that RHSP stimulates the Rep helicase unwinding reaction by stoichiometric binding to single-stranded DNA. However, a specific interaction between Rep protein and RHSP cannot be ruled out, since RHSP did not stimulate the duplex DNA unwinding reactions catalyzed by E. coli helicase I or the recently discovered 75-kDa helicase. RHSP did stimulate the duplex DNA unwinding reaction catalyzed by E. coli helicase II. The identification and subsequent purification of RHSP from cell extracts demonstrates the feasibility of using direct helicase assays to purify stimulatory proteins.     

A biochemical characterization of the adeno-associated virus Rep40 helicase

The human adeno-associated virus (AAV) has generated much enthusiasm as a transfer vector for human gene therapy. Although clinical gene therapy trials have been initiated using AAV vectors, much remains to be learned regarding the basic mechanisms of virus replication, gene expression, and virion assembly. AAV encodes four nonstructural, or replication (Rep), proteins. The Rep78 and Rep68 proteins regulate viral DNA replication, chromosomal integration, and gene expression. The Rep52 and Rep40 proteins mediate virus assembly. To better understand Rep protein function, we have expressed the Rep40 protein in Escherichia coli and purified it to near homogeneity. Like the other Rep proteins, Rep40 possesses helicase and ATPase activity. ATP is the best substrate, and Mg2+ is the most efficient divalent metal ion for helicase activity. A Lys to His mutation in the purine nucleotide-binding site results in a protein that inhibits helicase activity in a dominant negative manner. Rep40 unwinds double-stranded DNA containing a 3' single-stranded end, or blunt end, unlike the Rep68 and Rep52 enzymes, which have a strict requirement for DNA duplexes containing a 3' single-stranded end. Values for KATP in the ATPase assay are 1.1 +/- 0.2 mM and 1.2 +/- 0.2 mM in the absence and presence, respectively, of single-stranded DNA. Values for Vmax are 220 +/- 10 and 1,500 +/- 90 nmol/min/mg in the absence and presence, respectively, of single-stranded DNA. These studies provide the first enzymatic characterization of the AAV Rep40 protein and elucidate important functional differences between the AAV helicases.     

A two-site kinetic mechanism for ATP binding and hydrolysis by E. coli Rep helicase dimer bound to a single-stranded oligodeoxynucleotide.

Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reactions that are coupled to ATP binding and hydrolysis. We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep "P2S" dimer (formed with the single-stranded (ss) oligodeoxynucleotide, (dT)16), in which only one subunit of a Rep homo-dimer is bound to ssDNA. Pre-steady-state quenched-flow studies under both single turnover and multiple turnover conditions as well as fluorescence stopped-flow studies were used (4 degrees C, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 10 % (v/v) glycerol). Although steady-state studies indicate that a single ATPase site dominates the kinetics (kcat=17(+/-2) s-1; KM=3 microM), pre-steady-state studies provide evidence for a two-ATP site mechanism in which both sites of the dimer are catalytically active and communicate allosterically. Single turnover ATPase studies indicate that ATP hydrolysis does not require the simultaneous binding of two ATP molecules, and under these conditions release of product (ADP-Pi) is preceded by a slow rate-limiting isomerization ( approximately 0.2 s-1). However, product (ADP or Pi) release is not rate-limiting under multiple turnover conditions, indicating the involvement of a second ATP site under conditions of excess ATP. Stopped-flow fluorescence studies monitoring ATP-induced changes in Rep's tryptophan fluorescence displayed biphasic time courses. The binding of the first ATP occurs by a two-step mechanism in which binding (k+1=1.5(+/-0.2)x10(7) M-1 s-1, k-1=29(+/-2) s-1) is followed by a protein conformational change (k+2=23(+/-3) s-1), monitored by an enhancement of Trp fluorescence. The second Trp fluorescence quenching phase is associated with binding of a second ATP. The first ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-Pi). Binding of the second ATP then leads to the steady-state ATP cycle. As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to maintain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding. We propose that the roles of the two ATP sites may alternate upon binding DNA to the second subunit of the Rep dimer during unwinding and translocation using a subunit switching mechanism.     

A cellular single-stranded DNA-dependent ATPase associated with simian virus 40 chromatin

A single-stranded DNA-dependent ATPase from monkey kidney tissue culture cells (CV-1) has been found associated with SV40 chromatin. This ATPase activity is distinguishable from the ATPase activity of T-antigen by the following properties: the Km for ATP, elution from phosphocellulose, and stimulation of the ATPase activity by single-stranded DNA but not by double-stranded DNA. The ATPase has been isolated and characterized from the nuclei of uninfected cells. ATP hydrolysis is dependent on single-stranded DNA and a divalent cation. The km values for ATP and single-stranded DNA are 0.024 mM and 0.09 microgram/ml, respectively. The affinity of the ATPase for single-stranded DNA is sufficiently high that the enzyme co-sediments with single-stranded DNA in glycerol gradients. The binding of single-stranded DNA is independent of ATP and MgCl2; however, ATP hydrolysis increases the exchange of enzyme between different DNA molecules. Form I (superhelical) SV40 DNA is also a substrate for ATPase binding, but relaxed Form I, Form II (nicked circular), and double-stranded linear SV40 DNAs are not substrates. Because the DNA helix within chromatin is not under the same kind of tortional strain as Form I DNA, we hypothesize that the ATPase is bound to the single-stranded regions of replication forks in the SV40 chromatin.     

Properties of a 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.     

A Saccharomyces cerevisiae DNA helicase associated with replication factor C.

A novel DNA helicase has been isolated from Saccharomyces cerevisiae. This DNA helicase co-purified with replication factor C (RF-C) during chromatography on S-Sepharose, DEAE-silica gel high performance liquid chromatography (HPLC), Affi-Gel Blue-agarose, heparin-agarose, single-stranded DNA-cellulose, fast protein liquid chromatography MonoS, and hydroxyapatite HPLC. Surprisingly, the helicase could be separated from RF-C by sedimentation on a glycerol gradient in the presence of 200 mM NaCl. The helicase is probably a homodimer of a 60-kDa polypeptide, which by UV cross-linking has been shown to bind ATP. It has a single-stranded DNA-dependent ATPase activity, with a Km for ATP of 60 microM. The DNA helicase activity depends on the hydrolysis of NTP (dNTP), with ATP and dATP the most efficient cofactors, followed by CTP and dCTP. The DNA helicase has a 5' to 3' directionality and is only marginally stimulated by coating the single-stranded DNA with the yeast single-stranded DNA-binding protein RF-A.     

Enzyme-catalyzed DNA unwinding. A DNA-dependent ATPase from E. coli.

We have isolated a new DNA-dependent ATPase from E. coli. The enzyme has been purified to greater than 90% purity. It appears to be composed of two identical polypeptide chains of molecular weight 20,000. The enzyme catalyzed the hydrolysis of ATP in the presence, but not in the absence, of single-stranded DNA. Double-stranded DNA is not a cofactor. The products of hydrolysis are ADP and Pi. The enzyme also catalyzed strand separation of duplex DNA in the presence of ATP and E. coli DNA binding protein. Two E. coli proteins capable of promoting strand separation have been reported previously and have been termed helicase I and II (Abdel-Monem, M., and Hoffmann-Berling, H. (1977) Eur. J. Biochem. 79, 33-38). Accordingly, this protein is named helicase III.     

Enzymic unwinding of DNA. 1. Purification and characterization of a DNA-dependent ATPase from Escherichia coli.

Evidence from various sources in the literature suggests that, in connection with DNA, ATP dephosphorylation can be used to provide energy for mechanical effects. Starting from this concept we have studied a novel DNA-dependent ATPase purified to 90% homogeneity from Escherichia coli. The enzyme has a peptide weight near 180 000 and, in high salt, is a monomeric, probably highly anisometric molecule. In salt-free buffer, where the ATPase activity is highest, the enzyme forms aggregates. ATP is the preferred substrate (Km 0.27 mM) and dephosphorylated at the gamma-position at a maximal rate near 10(4) molecules per enzyme monomer per min at 35 degrees C. A requirement for divalent cation is best satisfied by Mg2+ or Ca2+ and the requirement for DNA best by the single-stranded, circular DNA of phages phiX174 (Km 62 nM nucleotide) and fd indicating that the enzyme recognizes internal DNA regions. When saturated with E. coli DNA unwinding protein phiX DNA is not accepted but, once in contact with the DNA, the enzyme is little inhibited by unwinding protein. Apparently the unwinding protein interferes preferentially with the recognition of DNA. The enzyme does not detectably cleave DNA, and for this and genetic reasons is not identical with the recBC ATPase or the K12 restriction ATPase of the extracted cells. The enzyme is probably not identical either with the dnaB-product-associated ATPase or the ATPase activity found in DNA polymerase III holoenzyme under appropriate conditions, and it is certainly not identical with a DNA-dependent ATPase of molecular weight 69 000 from E. coli which has recently been purified. Attempts to ascribe the enzyme to other genes, including recA, lex and rep, have failed.     

DNA-dependent adenosinetriphosphatase B from mouse FM3A cells has DNA helicase activity

We have detected at least four forms of DNA-dependent ATPase in mouse FM3A cell extracts [Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., & Yamada, M. (1984) Biochemistry 23, 529-533]. The purified fraction of one of the four forms, ATPase B, has been shown to have DNA helicase activity by using a DNA substrate which permits the detection of limited unwinding of the helix. The DNA substrate consists of single-stranded circular fd DNA and the hexadecamer complementary to the fd DNA, which bears an oligo(dT) tail at the 3' terminus. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.5 S on glycerol gradient centrifugation. The helicase required a divalent cation for activity (Mg2+ congruent to Mn2+ greater than Ca2+). The optimal concentrations of these divalent cations were 5 mM. The requirement of divalent cations of the DNA helicase activity was very similar to that for the DNA-dependent ATPase activity of ATPase B. The helicase activity was absolutely dependent on the presence of a nucleoside triphosphate. ATP was the most effective cofactor among the ribo- and deoxyribonucleoside triphosphates tested, and considerable levels of helicase activity were observed with other ribo- and deoxyribonucleoside triphosphates. The efficiency of a nucleoside triphosphate to serve as cofactor for the helicase activity correlated with the capacity of the nucleotide to serve as substrate for the DNA-dependent ATPase activity. The nonhydrolyzable ATP analogues such as adenosine 5'-O-(3-thiotriphosphate) were not effective for the helicase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-dependent adenosinetriphosphatase C1 from mouse FM3A cells has DNA helicase activity.

In our previous study, we identified four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, in mouse FM3A cells (Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., and Yamada, M. (1984) Biochemistry 23, 529-533). The DNA-dependent ATPase C1 has been purified and characterized in detail. A divalent cation and a polynucleotide cofactor were required for the ATPase activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Almost no ATPase activity was observed with S1 nuclease-treated native DNA. ATPase C1 hydrolyzed ATP only among the ribo- and deoxyribonucleoside triphosphates tested, and this fact distinguished ATPase C1 from ATPases B, C2, and C3, because the latter enzymes are capable of hydrolyzing both ATP and dATP. The purified DNA-dependent ATPase C1 fraction was shown to have a DNA helicase activity that was dependent on hydrolysis of ATP. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.2 S on glycerol gradient centrifugation. Both activities showed similar preferences for nucleoside 5'-triphosphates and similar requirements for divalent cations. The DNA helicase activity was inhibited by the addition of single-stranded DNAs that served as cofactor for the ATPase activity. The efficiency of a single-stranded DNA to inhibit DNA helicase activity correlated well with the capacity of the DNA to serve as cofactor for DNA-dependent ATPase activity. The helicase was shown to migrate along the DNA strand in the 5' to 3' direction, which is the same direction of migration of the mouse DNA helicase B (Seki, M., Enomoto, T., Yanagisawa, J., Hanaoka, F., and Ui, M. (1988) Biochemistry 27, 1766-1771).     

A prepriming DNA replication enzyme of Escherichia coli. I. Purification of protein n': a sequence-specific, DNA-dependent ATPase

Protein n', an enzyme essential for in vitro conversion of single-stranded phiX174 DNA to the duplex replicative form, has been purified about 16,000-fold from Escherichia coli. The enzyme is a single polypeptide chain with a native molecular weight of 76,000; about 70 enzyme molecules are present in an E. coli cell. Nearly homogeneous preparations display an ATPase (dATPase) activity which depends on a unique sequence in the phiX174 DNA. Replicative activity of n' protein and its phiX174 DNA-dependent ATPase activity were present in a constant ratio during the latter stages of purification, upon sedimentation in a glycerol gradient, and during heat inactivation. Further studies of the properties of protein n' are presented in a succeeding paper.     

Escherichia coli rep gene: sequence of the gene, the encoded helicase, and its homology with uvrD.

The sequence of a 2.67-kilobase section of the Escherichia coli chromosome that contains the rep gene has been determined. This gene codes for a protein of predicted Mr 72,800, a DNA helicase, which is also a single-stranded DNA-dependent ATPase. The sequenced region contains an open reading frame of the correct length and orientation to encode the Rep protein. A secondary structure for the protein can be formulated from the amino acid sequence. We have compared both the primary and the secondary structures of Rep with other proteins and find the greatest homology between Rep and E. coli helicase II, the product of the uvrD gene.     

An RNA-dependent ATPase from Chlamydomonas reinhardII

An RNA-dependent ATPase has been isolated from extracts of Chlamydomonas reinhardii. The enzyme catalyzes the hydrolysis of ATP, dATP, CTP and dCTP to the corresponding nucleoside diphosphate and Pi in the presence of Mg2+ or Mn2+ and an RNA cofactor. In 1 mM MgCl2 it displays the greatest activity with poly(A), poly(I) and poly(U); and somewhat lower activity with poly(C) and T7 RNA. Although the enzyme is active with single-stranded DNA, all the single-stranded RNAs tested were significantly more effective cofactors than any of the single or double-stranded DNAs tested. A comparison of this ATPase with other RNA-dependent ATPases indicates that is has more in common with the ATPase isolated from the nuclei of animal cells than with the RNA synthesis termination protein rho, the major RNA-dependent ATPase from Escherichia coli. Although chloroplasts of C. reinhardii are known to contain many bacterial-like gene expression components, the presence of an enzyme with close homology to the E. coli rho protein was not detected.     

Construction of a recombinase-deficient mutant recA protein that retains single-stranded DNA-dependent ATPase activity

The recA1 mutation is a single point mutation that replaces glycine 160 of the recA polypeptide with an aspartic acid residue. The mutant recA1 protein has a greatly reduced single-stranded DNA-dependent ATPase activity at pH 7.5 compared to the wild-type protein. Interestingly, the recA1 protein does exhibit a vigorous ATPase activity at pH 6.2. To explore the molecular basis of this pH effect, we used site-directed mutagenesis to replace aspartic acid 160 of the recA1 polypeptide with an isosteric, but nonionizing, asparagine residue. The new [Asn160]recA protein catalyzes ATP hydrolysis at pH 7.5 with the same turnover number as the wild-type protein. This result suggests that the activation of the recA1 protein ATPase activity that occurs at pH 6.2 may be due, in part, to neutralization of the negatively charged aspartic acid 160 side chain. Although it is an active single-stranded DNA-dependent ATPase, the [Asn160]recA protein is unable to complement a recA deletion in vivo and is unable to carry out the three-strand exchange reaction in vitro. Further examination of ATP hydrolysis (under strand exchange conditions) revealed that the ATPase activity of the [Asn160]recA protein is strongly suppressed in the presence of Escherichia coli single-stranded DNA-binding protein (a component of the strand exchange assay), whereas the ATPase activity of the wild-type recA protein is stimulated by the E. coli protein. To account for these results, we speculate that ATP may induce specific conformational changes in the wild-type recA protein that are essential to the DNA pairing process and that these conformational changes may not occur with the [Asn160]recA protein.     

Homologous pairing in genetic recombination. Purification and characterization of Escherichia coli recA protein

RecA protein, which is essential for genetic recombination in Escherichia coli, was extensively purified from a strain of E. coli which contained the recA gene cloned in a plasmid (Sancar, A., and Rupp, W. D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3144-3148). Using the DNA-dependent ATPase activity of recA protein as an assay, we obtained about 60 mg of purified recA protein from 100 g of cells. Ten micrograms or 1 microgram of the purified protein exhibited only one detectable band with Mr approximately = 40,000 upon sodium dodecyl sulfate-acrylamide gel electrophoresis. More than 99% of the ATPase activity of purified recA protein was dependent on single-stranded DNA. Purified recA protein had no detectable DNase, topoisomerase, or ligase activities. The enzyme was stable for a least a year when stored at 0-4 degrees C. The half-life of the ATPase activity of 25 microM recA protein was 37 min at 51 degrees C. Purified recA protein binds to single-stranded and double-stranded DNA, unwinds duplex DNA by a mechanism that is stimulated by single-stranded DNA or oligonucleotides, and pairs homologous single strands with duplex DNA.     

The rep mutation. VI. Purification and properties of the Escherichia coli rep protein, DNA helicase III.

The protein product of the rep gene of Escherichia coli is required for the replication of certain bacteriophage genomes (phi X174, fd, P2) and for the normal replication of E. coli DNA. We have used a specialized transducing phage, lambda p rep+, which complements the defect of rep mutants, to identify the rep protein. The rep protein has been purified from cells infected with lambda p rep+ phage; it has a molecular weight of about 70 000 and appears similar to the protein found in normal cells. Stimulation of phi X174 replicative form DNA synthesis in vitro was observed when highly purified rep protein was supplied to a cell extract derived from phi X-infected E. coli rep cells and supplemented with replicative form DNA. The purified protein has a single-stranded DNA-dependent ATPase activity and is capable of sensitizing duplex DNA to nucleases specific for single-stranded DNA. For this reason we propose the enzyme be called DNA helicase III. We infer that the rep protein uses the energy of hydrolysis of ATP to separate the strands of duplex DNA; the E. coli DNA binding protein need not be present. The rep3 mutant appeared to make a limited amount of active rep protein.     

DNA-dependent RNA polymerase from Pseudomonas aeruginosa

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having beta' (157,000), beta (148,000), sigma (87,000), and alpha 2 (45,000) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+, produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5-9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 degrees C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.     

Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).     

Purification and characterization of a DNA-dependent ATPase from Escherichia coli.

A DNA-dependent ATPase has been isolated and purified from an Escherichia coli cell-free extract. The ATPase has the following characteristics: preferential dependence on single-stranded DNA, specificity for ATP hydrolysis, Km value of 1.4 X 10-4 M for ATP, and molecular weight of approximately 69,000. The ATPase can be shown to bind to single stranded DNA. The resemblance between this ATPase and that isolated from vaccinia cores is discussed.