Mechanism of Inactivation of Transforming Deoxyribonucleic Acid by X Rays

Transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae was exposed to X rays either in phosphate buffer or in 10% yeast extract. Relations between determinations of biological inactivation, DNA uptake by competent H. influenzae, integration of DNA into the competent cell genome, and induced single-and double-strand breaks indicate that transforming DNA is inactivated by the direct and the indirect effect of X radiation primarily because integration of DNA is prevented as a result of the production of double-strand breaks.     

The transforming activity of sonicated Haemophilus influenzae DNA

Summary The inactivation of transforming Haemophilus influenzae DNA by sonication in aqueous solution was investigated. The molecular weight decrease of the molecules is the major factor in DNA inactivation. It impairs strongly the uptake of the DNA by the recipient bacteria, especially when the molecular weight is lower than 3x10⁶ daltons. The uptake of sonicated DNA by the bacterial cells seems not to be further reduced when molecules of about 0.5x10⁶ daltons are submitted to further depolymerisation. However the transforming activity of these molecules is still sensitive to further sonication. The transforming activity of the sonicated DNA is related in the last resort to the intact length of the DNA molecules, at the level of their single-strand structure, available for recombination. Rupture by ultrasound was found to be twice as efficient in reducing transforming activity as a nick induced by pancreatic DNAse.     

Biological properties of a Haemophilus influenzae restriction enzyme, Hind I.

A type I restriction enzyme from Haemophilus influenzae, Hind I, which requires adenosine 5' -triphosphate and 5-adenosyl methionine, was studied for its activity on transfecting and transforming deoxyribonculeic acid (DNA). The enzyme reduced the size of unmodified bacteriophage S2 DNA from 37 X 10(6) daltons to approximately 10 X 10(6) daltons, but did not affect modified S2 DNA. Unmodified transforming DNA was attacked in vitro by Hind I; however, relatively low levels of inactivation were obtained for single markers, and linked transformants were inactivated as a function of the distance between markers. In contrast, unmodified bacterial DNA was not inactivated in vivo for either single or linked markers by the Hind I restriction system, probably because the segments generated by Hind I were still capable of being integrated in vivo. The lack of preferential inactivation of markers by the enzyme suggests that it makes random breaks in the DNA.     

Degradation of transforming and transfecting DNA by the restriction endonucleases of type I and type II isolated from Haemophilus influenzae.

The restriction endonucleases of type I and II from Haemophilus influenzae were studied for their activity on transforming and transfecting DNA. Type I restriction enzyme from Haemophilus influenzae Rf, which requires adenosine 5'-triphosphate, reduced the size of unmodified bacterial DNA from 66x106 daltons to approximately 18x106 daltons and did not attack modified DNA. The action of this enzyme gives only a low level of inactivation of single and linked markers in the transforming DNA. In contrast the HP1c1 phage DNA was drastically inactivated by this enzyme. The endoR.Hind III degrades the ummodified bacterial DNA but the segments generated by this enzyme are still capable of being integrated in transformation. The enzyme has no activity on HP1c1 phage DNA.     

Uptake of heterologous DNA by Haemophilus influenzae.

With the use of highly competent Haemophilus influenzae cells, it was possible to demonstrate the uptake of heterologous DNAs. However, these DNAs, as expected, were only 1% or less as effective when competing for uptake with Haemophilus DNA. Escherichia coli DNA was removed from solution by competent cells to the extent expected if all the E. coli DNA particles contained at least one uptake recognition signal. The data were consistent with a model in which there was one uptake signal per 20 X 10(6) to 30 X 10(6) daltons of E. coli DNA. Since H. influenzae DNA has many more recognition signals, approximately one per 2 X 10(6) daltons (Danner et al., Gene 77:311-318, 1980; K. Vogt and S. H. Goodgal, submitted for publication), it has been suggested that the slower rate of E. coli DNA binding and the so-called specificity of Haemophilus DNA binding are due to the number of recognition signals per molecule of DNA as well as the nature of the DNA receptor (Vogt and Goodgal, submitted for publication). The specificity of native H. influenzae DNA binding does not apply to the uptake of denatured DNA in the transforming system (low pH) for denatured DNA.     

Integration of Deoxyribonuclease-Treated DNA in Bacillus subtilis Transformation

Normal preparations of B. subtilis DNA have weight average native molecular weights of 10 to 30 x 10⁶. For any given preparation the upper and lower 95% size limits may differ by a factor of ten or more. Single-stranded molecular weights indicate an average of 1 to 4 breaks per single strand of the native DNA. The reduction in transforming activity and viscosity following DNAase I digestion can be accounted for by a direct relationship between the transforming activity of a DNA and its single-stranded molecular weight. Uptake studies with DNAase I treated heavy (²H¹⁵N ³H) DNA show that single strand breaks inhibit integration less than transformation. A provisional estimate of the size of the integrated region based on correlating the single strand size of the donor-recipient complex with the donor-recipient density differences following alkali denaturation came to 1530 nucleotides. Using a competent, nonleaky thymine-requiring strain of B. subtilis grown in 5-BU medium before and after transformation, it was shown that (a) No detectable amount of DNA synthesis is necessary for the initial stages of integration, (b) Cells which have recently been replicating DNA are not competent. (c) Cells containing donor DNA show a lag in DNA replication following transformation, (d) When donor DNA is replicated it initially appears in a density region between light and hybrid. This indicates that it includes the transition point formed at the time of reinitiation of DNA synthesis in the presence of 5-BU following transformation. A model is proposed in which donor DNA is integrated at the stationary growing point of the competent cell, which is in a state of suspended DNA synthesis.     

Single-strand breakage on binding of DNA to cells in the genetic transformation of Diplococcus pneumoniae.

Mutants of Diplococcus pneumoniae that lack a membrane-localized DNAase are defective in transformation because entry of DNA into the cell is blocked. Such mutants still bind DNA on the outside of the cell. The bound DNA is double-stranded and its double-stranded molecular weight is unchanged. Its sedimentation behavior in alkali, however, shows that it has undergone single-strand breakage. The breaks are located randomly in both strands of the bound DNA at a mean separation of 2 × 10⁶ daltons of single-stranded DNA. Both binding and single-strand breakage occur in the presence of EDTA. Single-strand breaks are similarly formed on binding of DNA to normally transformable cells in the presence of EDTA. The single-strand breaks appear to be a consequence of attachment. DNA may be bound to the cell surface at the point of breakage.A mutant that is partially blocked in entry also binds DNA mainly on the outside of the cell. In the presence of EDTA, DNA bound by this mutant undergoes only single-strand breaks. In the absence of EDTA, however, double-strand breaks occur, apparently as a result of the initiation of entry. It is possible that the double-strand breaks arise from additional single-strand breaks opposite those that occurred on binding. The double-strand breaks presumably result from action of the membrane DNAase as it begins to release oligonucleotides from one strand segment while drawing the complementary strand segment into the cell.     

Loss of activity of transforming deoxyribonucleic acid after uptake by Haemophilus influenzae

Voll, Mary Jane (University of Pennsylvania, Philadelphia), and Sol H. Goodgal. Loss of activity of transforming deoxyribonucleic acid after uptake by Haemophilus influenzae. J. Bacteriol. 90:873-883. 1965.-Transforming deoxyribonucleic acid (DNA) which has been irreversibly removed from solution by competent cells undergoes a progressive loss in marker activity when tested by lysis of the cells and exposure to new recipient cells. The loss of activity is limited and marker-specific, with greater inactivation of those markers with lower efficiencies of transformation. Recipient factors or donor factors which have undergone recombination, as measured by the appearance of linked markers, do not undergo inactivation. The efficiency of transformation can be correlated with the sensitivity of a marker to inactivation after DNA uptake. A mutation which affects the efficiency of transformation is found to increase sensitivity to postuptake inactivation. The rate of inactivation is temperature-dependent. At temperatures of 20 and 45 C, marker inactivation can occur without concomitant recombination. During the uptake process, DNA is retained in an acid-insoluble form, indicating that the fate of Haemophilus influenzae DNA differs from the fate of transforming DNA in pneumococcus.     

Transformation and Deoxyribonucleic Acid Size: Extent of Degradation on Entry Varies with Size of Donor

The fate of label introduced as donor deoxyribonucleic acid (DNA) into competent cells of Diplococcus pneumoniae was determined immediately after entry at 25 C, as a function of the size of the donor DNA. Part of the label is found to be acid soluble, part has been incorporated into chromosomal DNA, apparently through reincorporation of degraded donor DNA, and part is found in single strands of length smaller than that of the input donor DNA strands. The last fraction apparently constitutes the precursor for integration of intact donor genetic markers and is referred to as the intact fraction. For large donor DNA the intact fraction contains over 80% of the total intracellular label, but the median strand length has been reduced to 2.2 x 10(6) daltons. For small donor molecules (1 x 10(5) to 6 x 10(5) daltons per strand) the fraction intact increases with donor size from 10 to 50% of the total intracellular label, and the median strand length of this fraction is half that of the donor strands. By combining these results with earlier data on the size dependence of the yield of transformants per unit of total intracellular donor label, we have calculated the probability that a marker in the intact fraction will be integrated, as a function of the length of the donor strand after entry. This probability has a linear dependence on strand length for activities below 40% of maximum, and extrapolates to zero activity at 77,000 daltons per strand.     

Repair of near-ultraviolet (365 nm)-induced strand breaks in Escherichia coli DNA. The role of the polA and recA gene products.

The action of near-ultraviolet (UV-365 nm) radiation in cellular inactivation (biological measurements) and induction and repair of DNA strand breaks (physical measurements) were studied in a repair-proficient strain and in polA-, recA-, uvrA-, and polA uvrA-deficient strains of Escherichia coli K-12. The induction of breaks in the polA and polA uvrA strains was linear with dose (4.0 and 3.7 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively). However, in the recA-, uvrA-, and repair-proficient strains, there was an initial lag in break induction at low doses and then a linear induction of breaks at higher doses with rates of 4.6, 2.8, and 3.2 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively. We interpret these strain differences as indicating simultaneous induction and repair of breaks in polymerase 1 (polA)-proficient strains under the 0 degrees C, M9 buffer irradiation conditions that, for maximum efficiency, require both the polA and recA gene products. Strand-break rejoining also occurred at 30 degrees C in complete growth medium. We propose that at least three (and possibly four) distinct types of pathways can act to reduce the levels of 365-nm radiation-induced strand breaks. A quantitative comparison of the number of breaks remaining with the number of lethal events remaining after repair in complete medium at 30 degrees C showed that between one and three breaks remain per lethal event in the wild-type and recA strains, whereas in the polA strain one order of magnitude more breaks were induced.     

Repair of MMS-induced DNA double-strand breaks in haploid cells of Saccharomyces cerevisiae, which requires the presence of a duplicate genome.

Summary The formation and repair of double-strand breaks induced in DNA by MMS was studied in haploid wild type and MMS-sensitive rad6 mutant strains of Saccharomyces cerevisiae with the use of the neutral and alkaline sucrose sedimentation technique. A similar decrease in average molecular weight of double-stranded DNA from 5–6x10⁸ to 1–0.7x10⁸ daltons was observed following treatment with 0.5% MMS in wild type and mutant strains. Incubation of cells after MMS treatment in a fresh drug-free growing medium resulted in repair of double-strand breaks in the wild type strain, but only in the exponential phase of growth. No repair of double-strand breaks was found when cells of the wild type strain were synchronized in G-1 phase by treatment with factor, although DNA single-strand breaks were still efficiently repaired. Mutant rad6 which has a very low ability to repair MMS-induced single-strand breaks, did not repair double-strand breaks regardless of the phase of growth.These results suggest that (1) repair of double-strand breaks requires the ability for single-strand breaks repair, (2) rejoining of double-strand breaks requires the availability of two homologous DNA molecules, this strongly supports the recombinational model of DNA repair.     

On the Nature of Recombinants Formed during Transformation in Hemophilus influenzae

During the process of transformation in Hemophilus influenzae integration of donor DNA, i.e. the formation of recombinant DNA, involves the incorporation of single-stranded DNA. Evidence was obtained from cesium chloride density gradient centrifugation of DNA from donor-recipient complexes that integration was accompanied by the formation of hybrid DNA with a density intermediate with respect to heavy, ²H, ¹⁵N, donor and light, ¹H, ⁴N recipient DNA. On denaturation the position of the heavy donor DNA moved closer to, but not all the way toward, the density position of the original donor DNA. In addition to supporting the idea of single-stranded incorporation, this evidence suggested that the integrated donor DNA was covalently linked to light recipient DNA. The DNA was taken up in the double-stranded form and no detectable amounts of denatured DNA could be found during the transformation process. However, during the process of integration an amount of donor atoms, equivalent to the amount of hybrid DNA formed, appeared in recipient DNA, and indicated that while one strand of DNA was integrated the other was broken down and resynthesized. The density of the hybrid DNA, as well as rebanding of denatured hybrid, indicated that the size of the integrated piece of DNA was large, approximately 6 x 10⁶ daltons.     

Characterization of Inducible Bacteriophages in Bacillus licheniformis

Two morphologically distinct and physically separable defective phages have been found in Bacillus licheniformis NRS 243 after induction by mitomycin C. One of them (PBLB) is similar to the defective phage PBSX of B. subtilis, which has a density of 1.373 g/cm(3) in CsCl and a sedimentation coefficient of 160S. PBLB incorporates into its head mainly bacterial deoxyribonucleic acid (DNA) which has a sedimentation coefficient of 22S and a buoyant density in CsCl of 1.706 g/cm(3). The other phage (PBLA) has a morphology similar to the temperate phage phi105 of B. subtilis; the head diameter is about 66 nm, and it possesses a long and noncontractile tail. PBLA has a density of 1.484 g/cm(3) in CsCl and the phage-specific DNA, which is exclusively synthesized after induction by mitomycin C, has a density of 1.701 g/cm(3). PBLA DNA is double-stranded and has a sedimentation coefficient of 36S, corresponding to a molecular weight of 34 x 10(6) to 35 x 10(6) daltons. The phage DNA has one interruption per single strand, giving single-stranded segments with molecular weights of 13 x 10(6) and 4 x 10(6) daltons. Common sequences between the two phage DNA species and with their host DNA have been demonstrated by DNA-DNA hybridization studies. Both phage particles kill sensitive bacteria. However, all attempts thus far to find an indicator strain to support plaque formation have been unsuccessful.     

Site-specific integration of Agrobacterium tumefaciens T-DNA via double-stranded intermediates

Agrobacterium tumefaciens-mediated genetic transformation involves transfer of a single-stranded T-DNA molecule (T strand) into the host cell, followed by its integration into the plant genome. The molecular mechanism of T-DNA integration, the culmination point of the entire transformation process, remains largely obscure. Here, we studied the roles of double-stranded breaks (DSBs) and double-stranded T-DNA intermediates in the integration process. We produced transgenic tobacco (Nicotiana tabacum) plants carrying an I-SceI endonuclease recognition site that, upon cleavage with I-SceI, generates DSB. Then, we retransformed these plants with two A. tumefaciens strains: one that allows transient expression of I-SceI to induce DSB and the other that carries a T-DNA with the I-SceI site and an integration selection marker. Integration of this latter T-DNA as full-length and I-SceI-digested molecules into the DSB site was analyzed in the resulting plants. Of 620 transgenic plants, 16 plants integrated T-DNA into DSB at their I-SceI sites; because DSB induces DNA repair, these results suggest that the invading T-DNA molecules target to the DNA repair sites for integration. Furthermore, of these 16 plants, seven plants incorporated T-DNA digested with I-SceI, which cleaves only double-stranded DNA. Thus, T-strand molecules can be converted into double-stranded intermediates before their integration into the DSB sites within the host cell genome.     

Transcription In Vitro by Reovirus-Associated Ribonucleic Acid-Dependent Polymerase

Digestion of purified reovirus type 3 with chymotrypsin degrades 70% of the viral protein and converts the virions to subviral particles (SVP). The SVP contain 3 of the 6 viral structural proteins and all 10 double-stranded ribonucleic acid (RNA) genome segments but not adenine-rich, single-stranded RNA. An RNA polymerase which is structurally associated with SVP transcribes one strand of each genome segment by a conservative mechanism in vitro. The single-stranded products include large (1.2 x 10(6) daltons), medium (0.7 x 10(6) daltons), and small (0.4 x 10(6) daltons) molecules which hybridize exclusively with the corresponding genome segments. The enzyme obtained by heating virions at 60 C synthesizes similar products. Kinetic and pulse-chase studies indicate that the different-sized products are synthesized simultaneously but at rates which are in the order: small > medium > large.     

Sequence-specific DNA uptake in transformation of Neisseria gonorrhoeae

Piliated, competent gonococci are known to preferentially take up homologous transforming DNA into the cell. We examined the mechanism for DNA uptake with pFA10, a hybrid 11.5-kilobase (kb) penicillin-resistant (Pcr) plasmid composed of heterologous DNA from a 7.2-kb Pcr plasmid and homologous DNA from a 4.2-kb gonococcal cryptic plasmid. The presence of the gonococcal cryptic plasmid DNA in the hybrid resulted in markedly increased transformation efficiencies in isogenic crosses as compared with the parent 7.2-kb Pcr plasmid. Uptake of 32P-end-labeled MspI or TaqI restriction fragments of the hybrid was limited to fragments entirely derived from the 4.2-kb gonococcal cryptic plasmid, indicating that DNA uptake was probably dependent on the presence of a specific DNA sequence. Since Haemophilus DNA did not inhibit transformation by the hybrid Pcr plasmid, the gonococcal DNA uptake sequence is different from the known sequence involved in homologous DNA uptake by Haemophilus spp.     

Single strand breakage and repair in eukaryotic DNA as assayed by S1 nuclease.

A sensitive new approach for measuring the repair of single strand breaks in DNA induced by low doses of gamma irradiation was tested in cultured fibroblasts from Chinese hamster lung, human afflicted with ataxia telangiectasia or Fanconi's anemia and in normal cells of early and late passages. The assay is based on the increasing rate of strand separation of DNA duplexes in alkali for molecules with increasing numbers of single strand scissions. DNA strand separation is shown to follow the relation, in F = -(1/Mn - const) - tbeta where F is the proportion of double-stranded DNA, detected as S1 nuclease resistant, after alkaline denaturation time, t. Mn is the number-average molecular weight of DNA between single strand breaks. beta less than 1 is an empirically determined constant. The results suggest an increase in the number-average molecular weight between breaks, Mn, with increasing times for repair. The final level attained corresponds to the Mn of control DNA in unirradiated cells. As few as one break introduced into 109 daltons of single-stranded control cell DNA can be detected. The kinetics, requirements and sensitivities of this assay are described.     

Morphology and Physical Properties of Staphylococcus Bacteriophage P11-M15

The Group B Staphylococcus phage P11-M15 is shown to be 51% protein and 49% deoxyribonucleic acid (DNA). The intact virion has a molecular weight of 66.7 x 10(6) daltons. The purified viral DNA has a molecular weight of 32.7 x 10(6) daltons. The intact virion is shown to be composed of a polyhedral head which is attached at one of its vertices to a flexible tail having helical symmetry. The tail structure is terminated by a complex baseplate which has sixfold symmetry. The virion contains a single molecule of double-stranded DNA which has no apparent single-strand nicks or single-stranded terminal redundancies.     

Dna Damage by Chemically Generated Singlet Oxygen

A naphthalenic endoperoxide was used as a non-photochemical source of singlet oxygen (¹O₂) to examine some interactions between this reactive oxygen species and DNA. High molecular weight DNA (ca. 10⁸ daltons) was exposed to 120 mol m^?31O₂ (cumulative concentration) and analyzed for interstrand crosslinkage by hydroxyl apatite chromatography following formamide denaturation. No evidence for ¹O₂-induced interstrand crosslinking was obtained. The capacity of ¹O₂ to generate strand breaks in single-stranded (ss) and double-stranded (ds) DNA was investigated by sucrose gradient centrifugation analysis of bacteriophage øX174 DNA. No direct strand breaks could be detected at neutral pH, whereas extensive strand breakage was observed after treatment with alkali. Possible biological consequences of ¹O₂ -exposure were assessed by examining the plaque-forming capacity of ss and ds øX 174 DNA molecules using wildtype Escherichia coli spheroplasts as recipients. Without any further treatment with heat or alkali, exposure to the endoperoxide resulted in a time- and dose-dependent inactivation, ss DNA being considerably more sensitive than ds DNA. From the present results and those reported earlier (Nieuwint et al.,²⁰) we infer that ¹O₂-induced inactivation of øX174 DNA is not due to DNA backbone breakage nor to interstrand crosslinking, but rather to some form of damage to the base or sugar moiety of the DNA, the exact nature of which remains to be elucidated.