Temperate bacteriophages of Streptococcus pneumoniae that contain protein covalently linked to the 5'' ends of their DNA.

We have characterized three temperate bacteriophages of pneumococcus (HB-3, HB-623, and HB-746). Although all the phages belong to the same family, the polypeptide composition of the virions and the DNA restriction endonuclease analysis of their DNAs revealed differences among the three phages. The genomes of these bacteriophages have been isolated as DNA-protein complexes. The protein is specifically associated with the two 5' termini of the DNA as shown by experiments carried out with exonucleases. The protein bound to the DNA in the three phages studied, iodinated in vitro with 125I, has a molecular weight of 23,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the complexes with chaotropic agents suggested that the protein is covalently bound to the 5' termini of the DNA. Comparative pulsed-field gel electrophoresis analysis and Southern hybridization of the SmaI restriction fragments of DNAs from one lysogenic bacteria and its parental strain revealed that the prophage genome was integrated in the host chromosome.     

Characterization of a protein covalently linked to the 5' termini of the DNA of Bacillus subtilis phage phi29.

We have isolated a covalent DNA-protein complex from bacteriophage φ29 particles. Polyacrylamide gel electrophoresis and tryptic peptide analysis showed that the protein present in the complex is very similar or identical to p3, an early induced protein essential for viral DNA replication.When the DNA-protein complex is treated with the restriction endonuclease EcoRI, the protein is specifically associated to the two terminal fragments, A and C. The protein is probably linked to the 5′ termini of the DNA since proteinase K-treated DNA is resistant to phosphorylation with polynucleotide kinase, even after treatment with alkaline phosphatase, while it is sensitive to exonuclease III. By electron microscopy the protein is visualized as a dot located at the ends of unit length DNA molecules.Mixed infection of Bacillus subtilis, at 42 °C, with ts² mutants in cistrons 2 and 3 only produces ts 2 progeny. This finding suggests that an inactive protein p3 bound to the DNA of the ts 3 mutant is not replaced by a functional protein and, as a consequence, replication of the ts 3 DNA does not occur.     

Analysis of the 5'-termini of nascent DNA chains synthesized in permeable cells of Bacillus subtilis

The nascent DNA synthesized by permeable cells of Bacillus subtilis in the presence of 5'-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP has been isolated and characterized. The newly synthesized DNA was isolated free from other cellular nucleic acids by affinity chromatography on thiol-substituted agarose. The number average chain length of the nascent DNA synthesized in one minute at 25 degrees C was 33 nucleotide residues, due to the chain-terminating action of 2',3'-dideoxyATP. Several lines of evidence indicated that at least 90% of the DNA thus isolated carried a terminally phosphorylated RNA moiety at its 5'-end: (1) the nascent DNA was resistant to exonucleolytic degradation by spleen phosphodiesterase unless first hydrolyzed by strong alkali or ribonuclease; (2) the 5'-termini of nascent DNA could not be phosphorylated by polynucleotide kinase unless first treated with alkaline phosphatase or subjected to hydrolysis by strong alkali or ribonuclease; (3) alkaline hydrolysis of nascent DNA labeled with 32P at the 5'-end released unlabeled DNA with a free 5'-terminus and 32P-labeled ribonucleoside 3',5'-bisphosphates; (4) ribonuclease degradation of similarly labeled material produced an unlabeled DNA-containing polynucleotide fraction and 32P-labeled ribo-oligonucleotides; (5) chromatography on dihydroxyboryl cellulose showed that the RNA moiety lacked a 3'-terminal cis-diol grouping (even after treatment with alkaline phosphatase) unless first subjected to the 3'-exonucleolytic action of bacteriophage T4 DNA polymerase. The sequence of the ribonucleotide chains was elucidated by end-group labeling with polynucleotide kinase and digestion with various ribonucleases. The ribonucleotide moiety was primarily three and four residues in length with the predominant sequence (pp)pApG(pC)1-2pDNA. The possibility that it represents a primer for discontinuous DNA synthesis is discussed.     

Identification of a protein linked to the ends of adenovirus DNA.

A DNA-protein complex from human adenoviruses has been further characterized by electron microscopy, radiochemical labeling and analytical ultracentrifugation. Preparations of the complex contain a large percentage of forms of DNA which are either circular or oligomeric and are readily distinguishable from preparations of pronase-treated adenovirus DNA by analytical ultracentrifugation in cesium chloride gradients containing 4 M guanidinium chloride. The protein component has been iodinated in vitro with ¹²⁵I using Bolton and Hunter reagent, and SDS-polyacrylamide gel analysis of the labeled protein indicates that it has an apparent molecular weight of 55,000 daltons. DNAase I digestion of the DNA-protein complex labeled with ³²PO₄ results in release of a ³²PO₄-labeled protein which remains labeled even after boiling in 1% SDS and 1% mercaptoethanol. Subsequent digestion of this entity with snake venom phosphodiesterase leads to release of ³²P₄-labeled 5′-phosphate deoxynucleotides. Digestion of the DNA-protein complex with Eco R1 and analysis of the isolated restriction fragments indicates that the protein is present on each terminal fragment. We conclude that there is a protein of 55,000 daltons directly attached to each 5′ end of molecule probably via a covalent linkage. We propose that the protein functions during DNA replication by facilitating priming of the progeny strands, thus allowing the 5′ ends of the DNA to be replicated.     

Origin and degradation of the RNA primers at the 5' termini of nascent DNA chains in Bacillus subtilis

We had earlier characterized the nascent DNA synthesized in permeable cells of Bacillus subtilis in the presence of 5-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP as being substituted at its 5' end with a ribonucleotide moiety of the sequence pApG(pC)1-2 DNA. In this paper, we examine the origin and turnover of the DNA-linked ribonucleotide and its relationship to DNA replication. At least 50% of the RNA-linked nascent DNA chains served as guanylate acceptors when incubated with GTP and the eukaryotic capping enzyme, indicating the presence of 5'-terminal di- or triphosphate groups and suggesting that the RNA moiety is synthesized de novo and is not a degradation product. In nascent DNA produced without limitation of chain growth by dideoxyATP, the degree of terminal ribonucleotide substitution was reduced by 50%, consistent with a linkage between RNA primer removal and DNA chain growth. Such a relationship was demonstrated directly by examining the RNA primer content of nascent DNA synthesized in the absence of dideoxyATP as a function of DNA chain length. As the DNA size increased from 40 to 200 nucleotide residues, the extent of RNA substitution declined from 80% to nearly 0%. Endgroup analysis showed that the loss of RNA was accompanied by a gradual shift from predominantly adenylate residues to 5'-terminal guanylate, consistent with a stepwise removal of ribonucleotides from the 5' end. Evidence that the nascent mercurated DNA synthesized under our experimental conditions was indeed a replicative intermediate came from the study of the time course of DNA chain growth and pulse-chase experiments. In the presence of the DNA ligase inhibitor NMN, mercurated DNA accumulated in two size classes with average length of approximately 750 and 8000 nucleotide residues, presumably representing the mature size of intermediates in discontinuous DNA synthesis. Comparison with the DNA size range at which the loss of the 5'-terminal RNA moiety occurred (40 to 200 residues) indicated that the processing of RNA primers occurred at an early stage during DNA chain elongation, and that moderate size intermediates in discontinuous DNA replication (greater than 200 nucleotides) have already lost their RNA primers.     

Efficient methods for attaching non-radioactive labels to the 5'' ends of synthetic oligodeoxyribonucleotides.

The syntheses are described of two types of linker molecule useful for the specific attachment of non-radioactive labels such as biotin and fluorophores to the 5' terminus of synthetic oligodeoxyribonucleotides. The linkers are designed such that they can be coupled to the oligonucleotide as a final step in solid-phase synthesis using commercial DNA synthesis machines. Increased sensitivity of biotin detection was possible using an anti-biotin hybridoma/peroxidase detection system.     

Conversion of sub-megasized DNA to desired structures using a novel Bacillus subtilis genome vector

A novel genome vector using the 4215 kb Bacillus subtilis genome provides for precise target cloning and processing of the cloned DNA to the desired structure. Each process highly dependent on homologous recombination in the host B.subtilis is distinguished from the other cloning systems. A 120 kb mouse jumonji (jmj) genomic gene was processed in the genome vector to give a series of truncated sub-megasized DNA. One of these truncated segments containing the first intron was copied in a plasmid by a recombinational transfer method developed for B.subtilis. DNA manipulation previously considered difficult is argued with respect to DNA size and accuracy.     

The relative rotation of the ends of Bacillus subtilis during growth

Observation of long single filaments of Bacillus subtilis 168 in depression slide cultures demonstrated that one end rotated relative to the other during growth. This was observed with suspended filaments, filaments attached to glass surfaces and single stranded filaments folded back on themselves growing as a double stranded helix. This extends Mendelson's 1976 conclusion to cases with no alternative interpretation to the hypothesis that as each cell grows, the structure of the peptidoglycan changes to rotate one end relative to the other.     

Characterization of the 5'' to 3'' exonuclease associated with Thermus aquaticus DNA polymerase.

Thermus aquaticus DNA polymerase was shown to contain an associated 5' to 3' exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novel in situ activity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2 or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5'-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5'-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5'-hydroxylated residue. Hydrolysis of a 5'-terminal residue from a nick was preferred threefold over the same 5'-end of duplex DNA. The 5' to 3' exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction.     

The 5'' ends of yeast killer factor RNAs are pppGp.

The 5' nucleotides of the double-stranded RNAs of yeast killer factor have been isolated by digestion with pancreatic, T1 and T2 RNase followed by two-dimensional electrophoresis. They were identified by bacterial alkaline phosphatase and snake venom phosphodiesterase digestions. Both the larger double-stranded RNA (L, of 2.5 x 10(6) daltons) and the smaller double-stranded RNA (M, of 1.4 x 10(6) daltons) have the 5' end groups pppGp. These 5' ends are dissimilar to those of the double-stranded RNAs of animal viruses but may be characteristic of the 5' ends of the double-stranded RNAs of fungal viruses.     

Sensitization by 5-Bromouracil of DNA in Bacillus subtilis Spores to Ionizing Radiation

Quantitative determination by fluorescence spectroscopy is possible because of the linear relationship between the intensity of emitted fluorescence and the fluorophore concentration. However, concentration quenching may cause the relationship to become nonlinear, and thus, the optimal dilution ratio has to be determined. In the case of fluorescence fingerprint (FF) measurement, fluorescence is measured under multiple wavelength conditions and a method of determining the optimal dilution ratio for multivariate data such as FFs has not been reported. In this study, the FFs of mixed solutions of tryptophan and epicatechin of different concentrations and composition ratios were measured. Principal component analysis was applied, and the resulting loading plots were found to contain useful information about each constituent. The optimal concentration ranges could be determined by identifying the linear region of the PC score plotted against total concentration.     

The initial attachment of transforming DNA to competent Bacillus subtilis.

Summary The initial attachment of transforming DNA to competent Bacillus subtilis is temperature independent between 25° and 45°. However, below 15° there is a significant reduction in the amount of DNA attached to competent cells. The DNA that is attached at 4° can lead to transformation or interfere effectively with the subsequent attachment of a distinctive DNA when the cells are shifted to a permissive temperature (37°). These data suggest that the attachment of DNA at 4° is to sites normally involved in the transformation process. The amount of DNA that is initially attached to the bacteria at 4° or 37° after perturbation of the cells by ionic strength changes, repetitive washings, or periodate oxidation varies with the temperature at which the treatment occurs. These results are consistent with a reorientation of the DNA attachment sites upon lowering the temperature to 4°, such that their affinity for DNA and susceptibility inhibitory treatments are reduced.National Institutes of Health Research Career Program Awardee, CA-K3-6487 during a portion of this investigation.     

Effect of 4,5'',8-trimethylpsoralen interstrand cross-links present in recipient Bacillus subtilis on the integration of transforming DNA.

When recipient Bacillus subtilis carrying chromosomal trimethylpsoralen cross-links were transformed, the donor marker activity decreased with the extent of cross-linking. Additional donor marker activity was lost upon incubation of the reextracted DNA with nuclease S1, particularly at higher levels of cross-linking. Physical analysis of the reextracted DNA showed that the donor DNA was progressively excluded from heteroduplex formation as the frequency of cross-links in the recipient DNA increased. In the donor-recipient complexes still being formed, increasing amounts of donor DNA became susceptible to nuclease S1 digestion under these conditions. These results suggest that resident interstrand cross-links interfere both with initiation of recombination and with the completion of heteroduplex formation.     

Nucleotide sequence of the cohesive single-stranded ends of Bacillus subtilis temperate bacteriophage phi 105.

The cohesive single-stranded ends of temperate Bacillus subtilis phage phi 105 were analyzed with the exonuclease activities of the Klenow fragment of DNA polymerase I and with exonuclease III and were found to be 3' extensions. Chemical sequencing of 3'-end-labeled fragments showed that the ends are 7-base extended 3' single strands and have the sequence: 5'-GCGCTCC-3'. 3'-CGCGAGG-5'     

Early stages in Bacillus subtilis transformation: association between homologous DNA and surface structures.

The addition of ethylenediaminetetraacetate to competent cultures of Bacillus subtilis irreversibly inhibited the transformability as well as the cellular binding of DNA. Our results show that the inhibition of DNA binding by ethylenediaminetetraacetate in whole cells, protoplasts, and membrane vesicles is mainly due to a permanent alteration of the DNA receptors. Transformation absolutely requires free magnesium ions, whereas DNA binding is a magnesium-independent step. In contrast to ethylenediaminetetraacetate, the absence of Mg2+ does not irreversibly affect the capacity of the competent cells to be transformed DNA-binding receptors located at the cell surface remain associated with the plasma membrane after protoplasting and after isolation of membrane vesicles. A Mg2+-dependent endonucleolytic activity associated with the membrane appears to be responsible for the lower levels of binding by protoplasts in the presence of this ion.     

Induction of DNA amplification in the Bacillus subtilis chromosome.

A system allowing the induction of DNA amplification in Bacillus subtilis was developed, based on a thermosensitive plasmid, pE194, stably integrated in the bacterial chromosome. An amplification unit, comprising an antibiotic resistance marker flanked by directly repeated sequences, was placed next to the integrated plasmid. Activation of pE194 replication led to DNA amplification. Two different amplification processes appeared to take place: one increased the copy number of all sequences in the vicinity of the integrated plasmid and was possibly of the onion skin type, while the other increased the copy number of the amplification unit only and generated long arrays of amplification units. These arrays were purified and shown to consist mainly of directly repeated amplification units but to also contain non-linear regions, such as replication forks and recombination intermediates. They were attached to the chromosome at one end only, and were, in general, not stably inherited, which suggests that they are early amplification intermediates. Longer arrays were detected before the shorter ones during amplification. When the parental amplification unit contained repeats which differed by a restriction site the arrays which derived thereof contained in a majority of cases only a single type of repeat. We propose that the amplified DNA is generated by rolling circle replication, and that such a process might underlie a number of amplification events.