Regulation of the synthesis of bacteriophage T4 gene 32 protein

The synthesis of T4 gene 32 product (P32) has been followed by gel electrophoresis of infected cell lysates. In wild-type infections, its synthesis starts soon after infection and begins to diminish about the time late gene expression commences. The absence of functional P32 results in a marked increase in the amount of the non-functional P32 synthesized. For example, infections of T4 mutants which contain a nonsense mutation in gene 32 produce the nonsense fragment at more than ten times the maximum rate of synthesis of the gene product observed in wild-type infections. All of the temperature-sensitive mutants in gene 32 that were tested also overproduce this product at the non-permissive temperature. This increased synthesis of the non-functional product is recessive, since mixed infections (wild-type, gene 32 nonsense mutant) fail to overproduce the nonsense fragment.Mutations in genes required for late gene expression (genes 33 and 53) as well as some genes required for normal DNA synthesis also result in increased production of P32. The overproduction in such infections is dependent on DNA synthesis; in the absence of DNA synthesis no overproduction occurs. This contrasts with the overproduction resulting from the absence of functional P32 which is not dependent on DNA synthesis.These results are compatible with a model for the regulation of expression of gene 32 in which the synthesis of P32 is either directly or indirectly controlled by its own function. Thus, in the absence of P32 function the expression of this gene is increased as is manifest by the high rate of P32 synthesis. It is further suggested that in infections defective in late gene expression and consequently in the maturation of replicated DNA, the increased P32 production is caused by the large expansion of the DNA pool. This DNA is presumed to compete for active P32 by binding it non-specifically to single-stranded regions, thus reducing the amount of P32 free to block gene 32 expression. Similarly, the aberrant DNA synthesized following infections with mutants in genes 41, 56, 58, 60 and 30, although quantitatively less than that produced in the maturation defective infections, can probably bind large quantities of P32 to single-stranded regions resulting in increased P32 synthesis.     

T4 gene 32 protein trypsin-generated fragments. Fluorescence measurement of DNA-binding parameters.

The intrinsic fluorescence of the T4 helix-destabilizing protein specified by gene 32 (32P) is not altered by the proteolytic removal of either the 6200-dalton COOH-terminal "A" region (32P*-A) or both the A and the 2300-dalton NH2-terminal "B" region (32P*-(A + B)). The intrinsic fluorescence of 32P, 32P*-A, and 32P*-(A + B) is decreased 23% by the addition of d(pT)8 and 34% by the addition of poly(dT). Saturation binding curves of the percentage of change in protein fluorescence as a function of nucleotide concentration show that the intact 32P as well as the two proteolysis-generated fragments all have association constants of approximately 10(6) M-1 for d(pT)8. This demonstrates that the DNA binding site is not contained within either the A or B regions of 32P. Both 32P and 32P*-A bind cooperatively to poly(dT) as evidenced by a 400- to 1000-fold increase in association constant for poly(dT) compared to d(pT)8. Since within the limits of our measurements 32P and 32P*-A bind equally well to poly(dT) (Kassoc approximately 5 . 10(8) M-1), the enhanced helix-destabilizing properties previously reported for 32P*-A cannot be accounted for by a significant increase in binding affinity of 32P*-A for single-stranded DNA. The binding constant for the 32P*-(A + B):poly(dT) complex is only 3-fold higher than that for the 32P*-(A + B):d(pT)8 complex, which confirms our proposal that the B region is essential for cooperative 32P:32P protein interactions.     

Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. II. Structural characterization of the covalent phi X A protein-DNA complex

In the preceeding paper (Brown, D. R., Roth, M. J., Reinberg, D., and Hurwitz, J. (1984) J. Biol. Chem. 259, 10545-10555), it was shown that following bacteriophage phi X174 (phi X) DNA synthesis in vitro using purified proteins, the phi X A protein could be detected covalently linked to nascent 32P-labeled DNA. This phi X A protein-[32P]DNA complex was the product of the reinitiation reaction. The phi X A protein-[32P]DNA complex could be trapped as a protein-32P-oligonucleotide complex by the inclusion of ddGTP in reaction mixtures. In this report, the structure of the phi X A protein-32P-oligonucleotide complex has been analyzed. The DNA sequence of the oligonucleotide bound to the phi X A protein has been determined and shown to be homologous to the phi X (+) strand sequence immediately adjacent (3') to the replication origin. The phi X A protein was directly linked to the 5' position of a dAMP residue of the oligonucleotide; this residue corresponded to position 4306 of the phi X DNA sequence. The phi X A protein-32P-oligonucleotide complex was exhaustively digested with either trypsin or proteinase K and the 32P-labeled proteolytic fragments were analyzed. Each protease yielded two different 32P-labeled peptides in approximately equimolar ratios. The two 32P-labeled peptides formed after digestion with trypsin (designated T1 and T2) and with proteinase K (designated PK1 and PK2) were isolated and characterized. Digestion of peptide T1 with proteinase K yielded a product which co-migrated with peptide PK2. In contrast, peptide T2 was unaffected by digestion with proteinase K. These results suggest that the phi X A protein contains two active sites that are each capable of binding covalently to DNA. The peptide-mononucleotide complexes T1-[32P]pdA and T2-[32P]pdA were isolated and subjected to acid hydrolysis in 6.0 N HCl. In each case, the major 32P-labeled products were identified as [32P] phosphotyrosine and [32P]Pi. This indicates that each active site of the phi X A protein participates in a phosphodiester linkage between a tyrosyl moiety of the protein and the 5' position of dAMP.     

Mechanism of the Inactivation of the Bacteriophage T1 in Aerosols

The mechanism of inactivation of bacteriophage T(1) in aerosols was studied by using (32)P-labeled phage. During inactivation, viability decreased in parallel with the adsorption of (32)P to the host, showing either that inactivated phage does not adsorb or that the deoxyribonucleic acid (DNA) has left the coat. A (32)P band at the density of free DNA was found when inactivated phage was analyzed in a CsCl gradient.     

Binding of homologous polymerized deoxyribonucleic acid by Streptomyces griseus

Germaine, G. R. (University of Minnesota, Minneapolis), and D. L. Anderson. Binding of homologous polymerized deoxyribonucleic acid by Streptomyces griseus. J. Bacteriol. 92:662-667. 1966-An irreversible P(32) deoxyribonucleic acid (DNA) binding system is described for Streptomyces griseus S104. The 69% guanine plus cytosine (GC) S. griseus DNA was bound by late exponential -early stationary phase cultures. Simultaneous addition of deoxyribonuclease with the P(32)-DNA to maximally receptive cultures reduced the uptake by 98%. Saturation of the binding ability occurred after 20-min incubation of cells with P(32)-DNA. Preparative cesium chloride density gradient centrifugation of the P(32)-DNA revealed that 98% of the label banded in the position expected for S. griseus DNA. Sedimentation of the P(32)-DNA at both 23 and 37 mug/ml indicated a molecular weight of 10.8 million. Attempts to transform S. griseus S104 auxotrophs to prototrophy by use of immediate and delayed selective procedures were unsuccessful.     

Specificity and kinetics defining the interaction between a murine monoclonal autoantibody and DNA

Interactions between a murine monoclonal anti-DNA autoantibody (BV17-45) and DNA were examined by direct binding and competitive radioimmunoassays. Binding isotherms constructed by titration of purified BV17-45 with a series of distinct 32P-labeled double-stranded DNA ([32P]dsDNA) fragments were super-impossible, suggesting: 1) BV17-45/[32P]dsDNA binding is independent of dsDNA size using fragments greater than or equal to 192 base pairs in length, and 2) BV17-45 does not exhibit stringent sequence specificity. Single-stranded DNA-specific monoclonal antibody BV04-01 did not react with [32P]dsDNA, confirming its duplex character. In competition experiments, BV17-45 cross-reacted with phage (phi X174, M13) RF AND VIRION DNAS AT PICOMOLAR concentrations. Selectivity for B-form DNA was suggested by the ability of poly(dA) . poly(dT), but not other helical duplex forms, to block BV17-45/[32P] dsDNA binding. Among the four deoxyribohomopolymers, only deoxyadenylic acid polymers completely inhibited BV17-45/[32P]dsDNA complex formation. [32P]dsDNA binding was relatively insensitive to ionic strength, suggesting minimal contribution of electrostatic forces to the binding free energy. Measured BV17-45/[32P]dsDNA association and dissociation rate constants (4 degrees C) were 7.4 X 10(6) M-1 s-1 and 9.2 X 10(-5) s-1, respectively, yielding a functional affinity of 8 X 10(10) M-1. Results are discussed in terms of the relative contribution of B-DNA structural and substructural determinants to the mechanism of BV17-45 recognition.     

Molecular nature of the mutations in gene ADE2 in Saccharomyces cerevisiae yeasts. III. Determination of the correlation of the GC AT pairs in genes ADE1 and ADE2

Lethal and mutagenic effects and the nature of mutations induced by decay of 32P incorporated into yeast cell DNA as 32P-deoxyguanosine monophosphate (32PdGMP) and 32P-thymidine monophosphate (32P-TMP), were studied. The lethal efficiency per 32P decay is independent of a labelled nucleotide incorporated into DNA. However, the mutagenic efficiency in ADE1, ADE2 genes per 32P decay is approximately 3 times greater for 32PdGMP than for 32P-TMP. This suggests that ADE1, ADE2 genes contain about 3 times more GC base pairs than AT pairs. Variations in a relative frequencies of GC leads to AT and AT leads to GC transitions were obtained depending upon a nucleotide labelled.     

Immunoprecipitation of SV40 replicating minichromosomes complexed with bacteriophage T4 gene 32 protein.

Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin. Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes. Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes. When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated. Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates. Protein analysis of the pelleted material revealed several proteins of viral and cellular origin. Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes. Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots. These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein. The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin.     

Parent-to-Progeny Transfer and Recombination of T4rII Bacteriophage

Transfer of parental, light (not substituted with 5-bromodeoxyuridine) (32)P-deoxyribonucleic acid (DNA) from rII(-) mutants of T4 bacteriophage to heavy (5-bromodeoxyuridine-substituted) progeny in Escherichia coli B was less homogeneous than in wild phages. The net transfer was 5 to 20% of the value for wild T4 phage, and the parental contribution per progeny DNA molecule amounted to 7 to 100% of the genome. Three classes could be distinguished, based on the density distribution of parental label in CsCl analysis of the progeny phages. "Far recombined" phages contain parental material only in semiconservatively replicated subunits covalently attached to progeny DNA, amounting to 5 to 10% parental contribution per genome. "Intermediate recombinants" contain, aside from conventional recombinant DNA, parental DNA banding at the original, light density. This DNA may be unattached to heavy progeny DNA or attached by weak bonds which are very sensitive to shearing during the extraction procedure. The parental contribution is 10 to 50% per progeny DNA molecule in this class. "Conservative" phages band close to the parental, light density in CsCl; their DNA is purely light. When the parental phage is labeled with both (3)H-leucine (capsid) and (32)P (DNA), the specific activity of (3)H/(32)P in the "conservative progeny" is 10 to 40% of that in the parental, showing that at least some of the (32)P in this area belongs to phages with parental DNA as the sole DNA component inside an unlabeled capsid, i.e., parental DNA which has been injected into the host and matured in a new capsid without replication or recombination. This phenomenon occurs to about the same extent in both single and multiple infection.     

A new and improved method for 3′-end labelling DNA using [α-32P]ddATP

We have synthesised dideoxyadenosine-5′-[α-³²P]triphosphate ([α-³²P]ddATP) at a specific activity of 3000 Ci/mmol and directly compared it with cordycepin-5′-[α-³²P]triphosphate ([α-³²P]KTP) as a means to 3′-end label DNA. The [α-³²P]ddATP was found to be three to five times more efficient than [α-³²P]KTP. Blunt and 3′-protruding ends were labelled more efficiently with [α-³²P]ddATP using terminal transferase than were the 5′-ends with [γ-³²P]ATP using polynucleotide kinase by standard methods. This improvement in efficiency of labelling DNA and the simplicity of the method allows 3′-end labelling of DNA to become a realistic alternative to 5′-end labelling. We have also compared [α-³²P]ddATP- and [α-³²P]KTP-labelled DNA in Maxam and Gilbert sequencing procedures and find that both give equally good results.     

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.     

Estimation of the growth rate of mixed ruminal bacteria from short-term DNA radiolabeling

A method based on 32P-labeling of DNA in short-term incubations was developed for estimating the growth rate of mixed rumen bacteria. A freeze/thaw procedure was optimized to quantitatively disrupt mixed rumen bacteria and extract bacterial DNA. The preliminary enzymatic lysis step, with lysozyme rather than proteinase K, sodium lauroyl sarcosine, and, to a lesser extent, sodium dodecyl sulfate (SDS) strongly improved cell disruption and DNA recovery rates. Sodium deoxycholate, CHAPS or Triton X-100 had no significant effect. Increasing the number of cycles or lowering the freezing temperature from -20 degrees C to -50 degrees C had no effect on DNA extraction efficiency while setting the thawing temperature at +60 degrees C rather than +37 degrees C slightly increased DNA yield but also increased its contamination with RNA. The method finally selected led to the lysis of at least 93% of cells and to the extraction of 85% of bacterial DNA. The kinetics of in vitro 32P incorporation into rumen bacteria DNA was then determined in batch incubations of strained rumen contents with no additional substrate. The curvilinear effects of the amount of 32P and the incubation time (5-15 min) on the DNA radioactivity were investigated by applying a Doehlert experimental design and fitting a second order polynomial model to data. The DNA radioactivity was linearly related to time (p<0.02) with other coefficients in the model being equal to zero (p>0.20). The incorporation of 32P into bacterial DNA was initiated approximately 70 s after the start of incubation. Taking into account the accuracy of scintillation counting, 10-15 min incubations, with 15 microCi 32P and 10 mL rumen contents per tube, appeared satisfactory for future studies.     

Inactivation of bacteriophages by decay of incorporated radioactive phosphorus

The inactivation of the phages T1, T2, T3, T5, T7, and lambda by decay of incorporated P(32) has been studied. It was found that these phages fall into two classes of sensitivity to P(32) decay: at the same specific activity of P(32) in their deoxyribonucleic acid (DNA), T2 and T5 are inactivated three times as rapidly as T1, T3, T7, and lambda. Since the strains of the first class were found to contain about three times as much total phosphorus per phage particle as those of the second) it appears that the fraction of all P(32) disintegrations which are lethal is very nearly the same in all the strains. This fraction alpha depends on the temperature at which decay is allowed to proceed, being 0.05 at -196 degrees C., 0.1 at +4 degrees C., and 0.3 at 65 degrees C. Decay of P(32) taking place only after the penetration of the DNA of a radioactive phage particle into the interior of the bacterial cell can still prevent the reproduction of the parental phage, albeit inactivation now proceeds at a slightly reduced rate. T2 phages inactivated by decay of P(32) can be cross-reactivated; i.e., donate some of their genetic characters to the progeny of a mixed infection with a non-radioactive phage. They do not, however, exhibit any multiplicity reactivation or photoreactivation. The fact that at low temperatures less than one-tenth of the P(32) disintegrations are lethal to the phage particle and the dependence of the fraction of lethal disintegrations on temperature can be accounted for by the double stranded structure of the DNA macromolecule.     

Characterization of nascent DNA fragments produced by excision of uracil residues in DNA.

Nascent short DNA chains could result from repair of incorporated uracil residues or be intermediates in discontinuous replication. We have characterized short DNA chains having apyrimidinic/apurinic-sites at 5' ends, the expected intermediates of repair, to distinguish them from RNA-linked replication intermediates. We have synthesized model substrates for the repair products; d(pRib[32P]poly(T)) and d(Rib[32P]poly(T)). Alkaline hydrolysis of both substrates has produced [5'-32P]poly(dT). Nascent short DNA was prepared from an Escherichia coli sof (dut) mutant, in this strain fragments from excision repair of uracil residues accumulate. The products of alkaline treatment are hardly digested by spleen exonuclease which selectively degrades 5'-hydroxyl-terminated DNA. These two results show that alkaline hydrolysis of the uracil repair fragments produces 5'-phosphoryl-terminated DNA, whereas it is known that 5'-hydroxyl-terminated DNA is generated from RNA-linked DNA molecules. The two types of nascent fragments thus can be distinguished by the 5'-terminal structure produced by an alkaline hydrolysis.     

Comparison of three nonradioactive and a radioactive DNA probe for the detection of target DNA by DNA hybridization

The specificity and sensitivity of three methods for the preparation and detection of nonradioactive probe DNA (biotin-nick translation, biotin-photolabel, and antigen-chemical linkage) were evaluated and compared with a nick-translated³²P-labeled DNA probe in DNA hybridization studies. The DNA probes were prepared from a restriction fragment (HindIII-3) from bacteriophage P1 DNA, and target DNA consisted of purified phage P1 DNA or P1 prophage DNA in lysogens ofEscherichia coli. A probe concentration of 50 ng/ml resulted in clear detection with the three nonradioactiveHindIII-3 DNA probes, whereas the specificity of the³²P-HindIII-3 DNA probe was satisfactory at a concentration of 25 ng/ml. However, the detection of false positives was greater with the³²P-labeled probe. The sensitivity of the radiolabeled DNA probe was marginally greater than that of the nonradioactive probes in dot blot hybridizations with purified phage P1 DNA. However, when the preparation time, ease of use, safety, duration of storage, and expense were compared for the four methods of labeling, the nonradiolabeled probes were generally superior to the radiolabeled probe.     

Nucleosomes from normal and regenerating rat liver

Micrococcal-nuclease digestion of rat liver nuclei selectively released mononucleosomes associated with ADP-ribosylated [Caplan, Ord & Stocken (1978) Biochem. J.174, 475-483] histone H1. Two classes of mononucleosome were detected, those that leaked out during digestion and those that were subsequently released by 5mm-sodium phosphate buffer (pH6.8)/0.2mm-NaEDTA. The former, from which histone H1 had been dissociated, contained 140-base-pair-length DNA and core histones;the latter contained core particles and mononucleosomes with histone H1 and 200-base-pair-length DNA. When normal liver nuclei were phosphorylated with [gamma-(32)P]ATP, dissociated histone H1, which could be separated from core particles with Sephadex G-200, showed (32)P uptake. (32)P uptake into histones H2A and poly(ADP-ribosyl)ated H3 was appreciable in core particles, but was less evident in nucleosomes still containing histone H1. When [(3)H]-thymidine was given to partially hepatectomized rats in S-phase, 5-10min pulses in animals of over 300g body wt. showed the presence of high-specific-radioactivity DNA in released core particles and mononucleosomes compared with DNA retained in the nuclear pellets. Mononucleosomes from rat livers in S-phase with new, [(3)H]lysine-containing histones, had higher (32)P incorporation in histones H1 and their core histones, than for di- or tri-nucleosomes. Thermal-denaturation properties of control and phosphorylated mononucleosomes and core particles were very similar; removal of histone H1 and non-histone chromosomal proteins in 0.5m-NaCl markedly increased the proportion of DNA ;melting' below 70 degrees C.     

Delta-elimination in the repair of AP (apurinic/apyrimidinic) sites in DNA.

[5'-32P]pdT8d(-)dT7, containing an AP (apurinic/apyrimidinic) site in the ninth position, and [d(-)-1',2'-3H, 5'-32P]DNA, containing AP sites labelled with 3H in the 1' and 2' positions of the base-free deoxyribose [d(-)] and with 32P 5' to this deoxyribose, were used to investigate the yields of the beta-elimination and delta-elimination reactions catalysed by spermine, and also the yield of hydrolysis, by the 3'-phosphatase activity of T4 polynucleotide kinase, of the 3'-phosphate resulting from the beta delta-elimination. Phage-phi X174 RF (replicative form)-I DNA containing AP (apurinic) sites has been repaired in five steps: beta-elimination, delta-elimination, hydrolysis of 3'-phosphate, DNA polymerization and ligation. Spermine, in one experiment, and Escherichia coli formamidopyrimidine: DNA glycosylase, in another experiment, were used to catalyse the first and second steps (beta-elimination and delta-elimination). These repair pathways, involving a delta-elimination step, may be operational not only in E. coli repairing its DNA containing a formamido-pyrimidine lesion, but also in mammalian cells repairing their nuclear DNA containing AP sites.     

Nucleosomal structure of Epstein-Barr virus DNA in transformed cell lines.

Micrococcal nuclease digestion was used to analyze Epstein-Barr virus (EBV) DNA structure in nuclei of transformed cells. Digests of virus-producing (P3HR-1), non-virus-producing (Raji), and superinfected Rajii cell nuclei were fractionated by electrophoresis on agarose gels, transferred to nitrocellulose, and hybridized to 32P-labeled EBV DNA. The viral DNA of Raji nuclei produced a series of bands on electrophoresis whose lengths were integral multiples of a unit size, which was the same as the repeat length of host DNA. Viral DNA in nuclei of P3HR-1 and superinfected Raji cells produced faintly visible bands superimposed on a smear of viral DNA which dominated the hybridization pattern. No differences were detected in the patterns when total DNA digests from Raji, P3HR-1, and an EBV DNA-negative cell line (U-698M) were analyzed by ethidium bromide staining or by hybridization with the use of 32P-labeled lymphoblastoid cell DNA as probe. We conclude that the EBV episomal DNA of Raji cells is folded into nucleosomes, whereas most of the viral DNA of P3HR-1 and superinfected Raji cells is not. This pattern of DNA organization differs signficantly from that in papova group viruses.     

Uncoupling of the DNA breaking and rejoining steps of Escherichia coli type I DNA topoisomerase. Demonstration of an active covalent protein-DNA complex

DNA topoisomerases have been shown to cleave DNA phosphodiester bond and simultaneously become linked to the DNA at the cleavage site via a phosphotyrosine linkage (Tse, Y.-C., Kirkegaard, K., and Wang, J. C. (1980) J. Biol. Chem. 255, 5560-5565). For prokaryotic DNA topoisomerases, this is observed only when denaturant or protease is added to the topoisomerase-DNA incubation mixture. Previous attempts to reform DNA phosphodiester bonds from the covalent protein-DNA complex have been unsuccessful. Using oligonucleotides as substrates, the cleavage reaction of Escherichia coli DNA topoisomerase I occurs spontaneously (Tse-Dinh, Y.-C., McCarron, B. G. H., Arentzen, R., and Chowdhry, V. (1983) Nucleic Acids Res. 11, 8691-8701). Upon reaction with oligo(dA) labeled with 32P using terminal transferase and [alpha-32P]dATP, the enzyme becomes covalently linked to the 32P-labeled oligonucleotide. This 32P label can then be transferred to the 3'-OH end of a linear or nicked duplex DNA molecule subsequently added to the reaction mixture. This phosphodiester bond rejoining reaction can occur at a recessed, blunt, or protruding 3'-end of double-stranded DNA. It requires magnesium ions. These observations suggest that the covalent protein-DNA complex is a true intermediate during topoisomerization. Implications on the structure of prokaryotic type I DNA topoisomerases as compared to their eukaryotic counterparts are discussed.     

State of hepatitis B viral DNA in a human hepatoma cell line.

PLC/PRF/5, a tissue culture cell line isolated from a human hepatocellular carcinoma and producing hepatitis B surface antigen, was studied for the presence of hepatitis B virus (HBV)-specific DNA and RNA. PLC/PRF/5 cell DNA accelerated the rate of reassociation of HBV [32P]DNA, and quantitative experiments indicated that the cells contained approximately four copies of viral DNA per haploid, mammalian cell DNA equivalent. PLC/PRF/5 DNA accelerated the rate of reassociation of all individual restriction endonucleases HincII and HaeIII fragments of HBV [32P]DNA, indicating that DNA from all regions of the viral genome is present in the cells. This suggests that these cells contain at least most, and possibly all, of the viral genome. Digestion of PLC/PRF/5 cell DNA with restriction endonuclease HindIII (an enzyme found not to cleave the DNA of any HBV isolate so far examined) yielded only three fragments, all larger than virion DNA, which contained HBV DNA base sequences, suggesting that HBV DNA is integrated in high-molecular-weight DNA at three different sites in these cells and that there is no viral DNA in an episomal form. PLC/PRF/5 cell [32P]RNA was found to hybridize with all restriction fragments of HBV DNA adequately tested, indicating that at least most, and possibly all, of the viral DNA in these cells is transcribed.