Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA

Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.     

Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA

The potent RNA polymerase inhibitors actinomycin D and 7-aminoactinomycin D are shown to bind to single-stranded DNAs. The binding occurs with particular DNA sequences containing guanine residues and is characterized by hypochromic UV absorption changes similar to those observed in interactions of the drugs with double-stranded duplex DNAs. The most striking feature of the binding is the dramatic (ca. 37-fold) enhancement in fluorescence that occurs when the 7-aminoactinomycin is bound to certain single-stranded DNAs. This fluorescence of the complex is also characterized by a 40-nm hypsochromic shift in the emission spectrum of the drug and an increase in the emission anisotropy relative to the free drug or the drug bound to calf thymus DNA. The fluorescence lifetimes change in the presence of the single-stranded DNA in a manner compatible with the intensity difference. Thus, there is an increase in the fraction of the emission corresponding to a 2-ns lifetime component compared to the predominant approximately 0.5-ns lifetime of the free drug. The 7-aminoactinomycin D comigrates in polyacrylamide gels with the single-stranded DNAs, and the fluorescence of the bound drug can be visualized by excitation with 540-nm light. The binding interactions are characterized by association constants of 2.0 x 10(6) to 1.1 x 10(7) M-1.     

Electron microscopic studies of bacteriophage M13 DNA replication.

Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation. The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis. In addition to single-stranded circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (theta, rolling circles), and, occasionally, RF DNAs with theta structures. The tails in theta molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of theta to other RF molecules does not change significantly with time after infection. The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion. This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map.     

Photodynamic guanine modification by hematoporphyrin is specific for single-stranded DNA with singlet oxygen as a mediator

Photodynamic modification of DNA by hematoporphyrin (Hp) was characterized by the DNA sequencing technique using 32P-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Mild photodynamic treatment of single-stranded DNA with Hp induced an alteration of guanine residues, and subsequent treatment with piperidine led to chain cleavages at each guanine residue. On the other hand, methylene blue plus light modified the guanine residues in both single-stranded and double-stranded DNA. ESR studies using 2,2,6,6-tetramethylpiperidine and 2,2,6,6-tetramethyl-4-piperidone as singlet oxygen traps demonstrated that Hp plus light produced almost the same amount of singlet oxygen as methylene blue plus light and that the photochemically generated singlet oxygen reacts significantly with guanylate but only slightly with other mononucleotides. An ESR spin destruction method revealed that photoexcited Hp generated porphyrin radical, but guanylate did not react with this radical. These results indicate that photoexcited Hp reacts with oxygen to generate singlet oxygen which oxidizes the guanine residues of single-stranded DNA and that the difference in photoreactivities of DNA with Hp and methylene blue may be explained in terms of the structural difference in their intercalating abilities.     

Ring-opened 7-methylguanine residues in DNA are a block to in vitro DNA synthesis.

Single-stranded M13mp18 phage DNA was methylated with dimethylsulfate (DMS), and further treated with alkali to ring-open N7-methylguanine residues and yield 2-6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) residues. Nucleotide incorporation during in vitro DNA synthesis on methylated template using E. coli DNA polymerase Klenow fragment (Kf polymerase) was reduced compared to the unmethylated template. Additional treatment of the methylated template with NaOH to generate Fapy residues, further reduced in vitro DNA synthesis compared to the synthesis on methylated templates, which suggested that Fapy residues were a block to in vitro DNA synthesis. Analysis of the termination products on sequencing gels, assuming that synthesis stops one base before a blocking lesion, indicated that arrest of DNA synthesis upon direct alkylation of single-stranded DNA occurred 1 base 3' to template adenine residues in the case of Kf polymerase and 1 base 3' to adenine and cystosine residues for T4 polymerase. When the alkylated templates were treated with NaOH to produce a template which converted all the N7-methylguanine residues to Fapy residues, the blocks to DNA synthesis were still observed one base before adenine residues. In addition to the stops previously observed for the methylated templates, however, new stops occurred one base 3' to template guanine residues for synthesis using both Kf polymerase and T4 polymerase. Fapy residues, therefore, represent a potential lethal lesion which may also arrest in vivo DNA synthesis if not repaired.     

Replication of parvoviral DNA. I. Characterization of a nuclear lysate system.

We have developed a nuclear lysate system from infected, synchronized cells capable of synthesizing unit-length parvoviral DNA in vitro. It was necessary to supplement the nuclear lysates with the polyamines, spermidine and spermine, to prevent degradation of template and product DNAs. In this system RF, RI, and single-stranded progeny DNAs were synthesized. Label incorporated in viral RF DNA in vivo appeared first in RI DNA and then in single-stranded DNA during incubation in vitro. By sedimentation the product DNAs were identical to those found in infected cells. Their viral identity was confirmed by hybridization. The addition of ribonucleotides, RNase, or alpha-amanitin did not affect parvoviral DNA synthesis in this system. The results with the specific inhibitors of mammalian DNA polymerases, aphidicolin, N-ethylmaleimide, and 2',3'-dideoxythymidine 5'-triphosphate indicated that DNA polymerase alpha was required for synthesis of parvoviral DNA in the nuclear lysates. This requirement was confirmed by experiments with antibody to bovine DNA polymerase alpha.     

Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase

Studies on the composition and characterization of DNA product(s) synthesized by calf thymus terminal deoxynucleotidyl transferase were performed using homopolymeric single-stranded, calf thymus double-stranded, and native DNA resident in calf thymus chromatin preparations as priming DNA species. Synthesis was carried out using equimolar concentrations of all four deoxynucleoside triphosphates as substrates and Mg²⁺ or Mn²⁺ as an effective divalent cation. Irrespective of the nature of the priming DNA or the divalent cation, the DNA product contained 60–70% dGMP residues, 10–15% each of the two pyrimidine residues, and 5–10% dAMP residues. The product synthesized using chromatin DNA as initiator was predominantly single-stranded and its synthesis was resistant to actinomycin D. The predilection of terminal deoxynucleotidyl transfease to synthesize dGMP-rich products on natural or homopolymeric DNA primers suggests that such products may represent biologically important recognition signal sequences.     

Spin-labelling of DNA with hydrazine mustard spin label (HMSL)

1. The hydrazine mustard spin label (HMSL), recently synthesized in our laboratory (Raikova, 1977) was used for spin-labelling of DNA. 2. It alkylates both double- and single-stranded DNAs. 3. The reaction of HMSL with DNA was studied with respect to the kinetics of alkylation, dependence on salt concentration and base specificity. 4. It was found that HMSL is a base-specific reagent, alkylating preferentially guanine. According to their ability to bind HMSL, the four deoxyribonucleotides are ordered in the following way: G greater than A greater than C greater than T. 5. The EPR spectra obtained strongly depended on the secondary structure of the spin-labelled DNA: unlike the immobilized spectra of the double-stranded DNAs (2AZZ = 44.8G), the EPR spectra of single-stranded DNAs were non-immobilized (2AZZ = 32.8 G). 6. When sheared double-stranded DNA was spin-labelled, the parameters of the EPR spectrum depended also on the GC content of DNA.     

Biological properties of imidazole ring-opened N7-methylguanine in M13mp18 phage DNA.

Guanine residues methylated at the N-7 position (7-MeGua) are susceptible to cleavage of the imidazole ring yielding 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7-MeGua). The presence of Fapy-7-MeGua in DNA template causes stops in DNA synthesis in vitro by E. coli DNA polymerase I. The biological consequences of Fapy-7-MeGua lesions for survival and mutagenesis were investigated using single-stranded M13mp18 phage DNA. Fapy-7-MeGua lesions were generated in vitro in phage DNA by dimethylsulfate (DMS) methylation and subsequent ring opening of 7-MeGua by treatment with NaOH (DMS-base). The presence of Fapy-7-MeGua residues in M13 phage DNA correlated with a significant decrease in transfection efficiency and an increase in mutation frequency in the lacZ gene, when transfected into SOS-induced JM105 E.coli cells. Sequencing analysis revealed unexpectedly, that mutation rate at guanine sites was only slightly increased, suggesting that Fapy-7-MeGua was not responsible for the overall increase in the mutagenic frequency of DMS-base treated DNA. In contrast, mutation frequency at adenine sites yielding A----G transitions was the most frequent event, 60-fold increased over DMS induced mutations. These results show that treatment with alkali of methylated single-stranded DNA generates a mutagenic adenine derivative, which mispairs with cytosine in SOS induced bacteria. The results also imply that the Fapy-7-MeGua in E. coli cells is primarily a lethal lesion.     

Chemical Methylation of RNA and DNA Viral Genomes as a Probe of In Situ Structure

We used [methyl-(3)H] dimethyl sulfate to probe the genome structures of several RNA and DNA viruses. We compared sites of modification in nucleic acids that were methylated chemically before and after extraction from purified virions. With both single-stranded and double-stranded substrates alkylation occurred mainly at the N7 position of guanine. However, adenine N1 atoms were differentially accessible in single-stranded RNA and DNA. For example, the ratios of 1-methyladenosine to 7-methylguanosine for reovirus mRNA and deproteinized genome RNA were 0.43 and 0.03, respectively. Members of the Reoviridae methylated in situ yielded RNAs with ratios of 0.04 to 0.08, indicating that the intravirion genomes were double stranded. We obtained ratios of 0.26 and 0.35 for the RNAs of dimethyl sulfate-treated brome mosaic and avian sarcoma virions, respectively, which was consistent with partial protection of adenine N1 sites by structural proteins or genome conformation or both. The ratios of 1-methyladenosine to 7-methylguanosine for vaccinia virus DNAs methylated in situ (0.10) and after phenol extraction (0.14) were less than the ratios for phiX174 and M13 DNAs (0.39 to 0.64) but considerably greater than the ratio observed with adenovirus DNA (0.002 to 0.02). The presence of a single-stranded region(s) in the vaccinia virus genome was confirmed by S1 nuclease digestion of [methyl-(3)H] DNA; the released radiolabeled fraction had a ratio of 0.41, compared with 0.025 for the residual duplex DNA. In addition to the structure-dependent accessibility of adenine N1, methylation of adenine N3 was severalfold lower in the intravirion genomes of vaccinia virus, phiX174, and adenovirus than in the corresponding extracted DNAs. Chemical methylation of virions and subviral particles should be useful for in situ analyses of specific regions of RNA and DNA genomes, such as the sites of protein binding during virus maturation.     

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2).     

Mechanism of site-specific DNA damage induced by ozone

Ozone has been shown to induce lung tumors in mice. The reactivity of ozone with DNA in an aqueous solution was investigated by a DNA sequencing technique using 32P-labeled DNA fragments. Ozone induced cleavages in the deoxyribose-phosphate backbone of double-stranded DNA, which were reduced by hydroxyl radical scavengers, suggesting the participation of hydroxyl radicals in the cleavages. The ozone-induced DNA cleavages were enhanced with piperidine treatment, which induces cleavages at sites of base modification, but the inhibitory effect of hydroxyl radical scavengers on the piperidine-induced cleavages was limited. Main piperidine-labile sites were guanine and thymine residues. Cleavages at some guanine and thymine residues after piperidine treatment became more predominant with denatured single-stranded DNA. Exposure of calf thymus DNA to ozone resulted in a dose-dependent increase of the 8-oxo-7,8-dihydro-2'-deoxyguanosine formation, which was partially inhibited by hydroxyl radical scavengers. ESR studies using 5,5-dimethylpyrroline-N-oxide (DMPO) showed that aqueous ozone produced the hydroxyl radical adduct of DMPO. In addition, the fluorescein-dependent chemiluminescence was detected during the decomposition of ozone in a buffer solution and the enhancing effect of D2O was observed, suggesting the formation of singlet oxygen. However, no or little enhancing effect of D2O on the ozone-induced DNA damage was observed. These results suggest that DNA backbone cleavages were caused by ozone via the production of hydroxyl radicals, while DNA base modifications were mainly caused by ozone itself and the participation of hydroxyl radicals and/or singlet oxygen in base modifications is small, if any. A possible link of ozone-induced DNA damage to inflammation-associated carcinogenesis as well as air pollution-related carcinogenesis is discussed.     

Fapyadenine is a moderately efficient chain terminator for prokaryotic DNA polymerases

Hypoxanthine?xanthine oxidase?Fe3+?ethylenediaminetetraacetate (EDTA) was used to modify ss M13 mp18 phage DNA. The dominant base modifications found by GC/IDMS-SIM were FapyGua, FapyAde, 8-hydroxyguanine, and thymine glycol. Analysis of in vitro DNA synthesis on oxidatively modified template by three DNA polymerases revealed that T7 DNA polymerase and Klenow fragment of polymerase I from Escherichia coli were blocked mainly by oxidized pyrimidines in the template whereas some purines that were easily bypassed by the prokaryotic polymerases constituted a block for DNA polymerase beta from calf thymus. DNA synthesis by T7 polymerase on poly(dA) template, where FapyAde content increased 16-fold on oxidation, yielded a final product with a discrete ladder of premature termination bands. When DNA synthesis was performed on template from which FapyAde, FapyGua, and 8OHGua were excised by the Fpg protein new chain terminations at adenine and guanine sites appeared or existing ones were enhanced. This suggests that FapyAde, when present in DNA, is a moderately toxic lesion. Its ability to arrest DNA synthesis depends on the sequence context and DNA polymerase. FapyGua might possess similar properties.     

Complex formation between p53 and replication protein A inhibits the sequence-specific DNA binding of p53 and is regulated by single-stranded DNA.

Human replication protein A (RP-A) (also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. Potentially important to both these functions, it is also capable of complex formation with the tumor suppressor protein p53. Here we show that although p53 is unable to prevent RP-A from associating with a range of single-stranded DNAs in solution, RP-A is able to strongly inhibit p53 from functioning as a sequence-specific DNA binding protein when the two proteins are complexed. This inhibition, in turn, can be regulated by the presence of various lengths of single-stranded DNAs, as RP-A, when bound to these single-stranded DNAs, is unable to interact with p53. Interestingly, the lengths of single-stranded DNA capable of relieving complex formation between the two proteins represent forms that might be introduced through repair and replicative events. Increasing p53 concentrations can also overcome the inhibition by steady-state levels of RP-A, potentially mimicking cellular points of balance. Finally, it has been shown previously that p53 can itself be stimulated for site-specific DNA binding when complexed through the C terminus with short single strands of DNA, and here we show that p53 stays bound to these short strands even after binding a physiologically relevant site. These results identify a potential dual role for single-stranded DNA in the regulation of DNA binding by p53 and give insights into the p53 response to DNA damage.     

Site-specific oxidation at GG and GGG sequences in double-stranded DNA by benzoyl peroxide as a tumor promoter

Benzoyl peroxide (BzPO), a free-radical generator, has tumor-promoting activity. As a method for approaching the mechanism of tumor promoter function, the ability of oxidative DNA damage by BzPO was investigated by using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. BzPO induced piperidine-labile sites at the 5'-site guanine of GG and GGG sequences of double-stranded DNA in the presence of Cu(I), whereas the damage occurred at single guanine residues of single-stranded DNA. Both methional and dimethyl sulfoxide (DMSO) inhibited DNA damage induced by BzPO and Cu(I), but typical hydroxyl radical ((*)OH) scavengers, superoxide dismutase (SOD) and catalase, did not inhibit it. On the other hand, H(2)O(2) induced piperidine-labile sites at cytosine and thymine residues of double-stranded DNA in the presence of Cu(I). Phenylhydrazine, which is known to produce phenyl radicals, induced Cu(I)-dependent damage at thymine residues but not at guanine residues. These results suggest that the BzPO-derived reactive species causing DNA damage is different from (*)OH and phenyl radicals generated from benzoyloxyl radicals. BzPO/Cu(I) induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in double-stranded DNA more effectively than that in single-stranded DNA. Furthermore, we observed that BzPO increased the amount of 8-oxodG in human cultured cells. Consequently, it is concluded that benzoyloxyl radicals generated by the reaction of BzPO with Cu(I) may oxidize the 5'-guanine of GG and GGG sequences in double-stranded DNA to lead to 8-oxodG formation and piperidine-labile guanine lesions, and the damage seems to be relevant to the tumor-promoting activity of BzPO.     

Electron microscopy of single-stranded structures in the DNA of competent Haemophilus influenzae cells.

Chromosomal DNAs from exponential-phase and competent cells of Haemophilus influenzae were examined by electron microscopy to determine whether the chromosome undergoes structural changes during competence development. Single-stranded gaps and single-stranded tails formed in chromosomal DNA during competence development. The generation of gaps was dependent on the rec-2 function. Since the rec-2 mutant is defective in the translocation of donor DNA, it was inferred that the gaps were involved in the translocation step of transformation. The generation of single-stranded tails was independent of the rec-1 and rec-2 genes. Therefore, these structures were assumed to play no direct role in the interaction of donor and recipient DNAs during transformation. Gaps were preferentially associated with a readily denaturable, possibly A + T-rich fraction of the genome. This finding raised the possibility that hot spots for transformation might be associated with A + T-rich DNA.     

Micro- and nanoparticles of condensed DNA Formed in PCR with <Emphasis Type="Italic">Taq</Emphasis> polymerase and plasmid DNA as a template

Formation of micro- and nanoparticles of condensed DNA during PCR with microbial genomic DNA or plasmid DNA as templates was reported previously. Initially, the microparticles were formed using a thermostable KlenTaq polymerase, which is a deletion variant of Taq polymerase. The present work shows that Taq polymerase is also capable of efficient formation of micro- and nanoparticles of condensed DNA in PCR. Electron microscopy revealed a number of morphological types (more than four) of microparticles produced in PCR with different reaction buffers in the presence of Taq polymerase and different plasmid DNAs as a template. In the case of some kinds of amplicons, an increase in the number of thermal cycles was shown to result in production of numerous nanowires and electron-dense spherical nanoparticles. The PCR conditions for preferential formation of discs (or ellipsoids) a few micrometers in diameter and several dozens of nanometers in thickness were determined. The structure of microparticles formed in the presence of Taq polymerase was found to depend on the level of synthesis of single-stranded DNA fragments in PCR. Experiments with nuclease S1 revealed that, along with double-stranded DNAs of the amplicon, micro- and nano-particles contained single-stranded DNA fragments, which were absolutely necessary for their formation. In light of these data, the molecular mechanism of micro- and nanoparticle formation in the course of PCR is discussed.     

Methylglyoxal, an endogenous aldehyde, crosslinks DNA polymerase and the substrate DNA

Methylglyoxal, a known endogenous and environmental mutagen, is a reactive α-ketoaldehyde that can modify both DNA and proteins. To investigate the possibility that methylglyoxal induces a crosslink between DNA and DNA polymerase, we treated a ‘primed template’ DNA and the exonuclease-deficient Klenow fragment (KF^exo–) of DNA polymerase I with methylglyoxal in vitro. When the reaction mixtures were analyzed by SDS–PAGE, we found that methylglyoxal induced a DNA–KF^exo– crosslink. The specific binding complex of KF^exo– and ‘primed template’ DNA was necessary for formation of the DNA–KF^exo– crosslink. Methylglyoxal reacted with guanine residues in the single-stranded portion of the template DNA. When 2′-deoxyguanosine was incubated with Nα-acetyllysine or N-acetylcysteine in the presence of methylglyoxal, a crosslinked product was formed. No other amino acid derivatives tested could generate a crosslinked product. These results suggest that methylglyoxal crosslinks a guanine residue of the substrate DNA and lysine and cysteine residues near the binding site of the DNA polymerase during DNA synthesis and that DNA replication is severely inhibited by the methylglyoxal-induced DNA–DNA polymerase crosslink.