Benzo(a)pyrene 7,8-dihydrodiol-9,10-oxide modification of DNA: Relation to chromatin structure and reconstitution

Purified duck reticulocyte DNA was incubated in vitro with a 7,8-dihydrodiol-9,10-oxide derivative of benzo(a)pyrene (BPDE). The carcinogen-modified DNA was somewhat more susceptible to partial digestion by the single strand specific endonuclease S₁ than unmodified DNA, suggesting slight denaturation of the helix at sites of modification. Chromatin was reconstituted in vitro utilizing this carcinogen-modified DNA and unmodified-chromatin associated proteins. This reconstituted chromatin showed the same kinetics and extent of digestion by Staphylococcal nuclease, and similar nucleosome profiles on sucrose density gradient centrifugation, as those obtained with native chromatin or chromatin reconstituted with unmodified DNA. Moreover, polyacrylamide gel electrophoresis of DNA fragments obtained from nuclease digests gel electrophoresis of DNA fragments obtained from nuclease digests of the reconstituted chromatins suggested that the chromatin containing carcinogen-modified DNA had the same subnucleosome structure as that reconstituted with unmodified DNA. In a separate set of studies intact duck reticulocyte chromatin was reacted directly with BPDE. Nuclease digestion studies indicated that 65% of the carcinogen was bound to the ‘open’ regions of chromatin, and 35% to ‘closed’ regions.These results indicate that although covalent binding of a benzo(a)pyrene (BP) derivative to DNA produces local distortions in conformation of the helix, this modification does not appear to interfere with the ability of the DNA to associate with histones to form nucleosome structures. In addition, although DNA in the open regions of chromatin is more susceptible to reaction with the BP derivative, there is appreciable reaction with the DNA associated with histones.     

Thermal denaturation of DNA in the chromatin fragments released by mild micrococcal nuclease digestion of HTC cell nuclei

In chromatin a minor fraction melts at a temperature lower than deproteinized DNA, which may be assigned to DNA destabilizing proteins. We attempted to localize the destabilized DNA in the various chromatin fragments separated by electrophoresis after a mild micrococcal nuclease digestion. The small released fragments are enriched in coding sequences. About 20% of the DNA extracted from the released nucleosomes are single-stranded, 60% of the DNA in these fragments are digested by nuclease S₁ after incubation at low temperature, which suggests that the DNA destabilizing proteins are present in the released nucleosomes. Hybridization studies have shown that 25% of the DNA in nucleosomes are specific of this class of fragments. DNA destabilizing proteins could be associated with the specific sequences.     

An alternative view of mammalian DNA sequence organization: II. Short repetitive sequences are organized into scrambled tandem clusters in Syrian hamster DNA

Hyperchromicity, S₁ nuclease digestion, and reassociation studies of Syrian hamster repetitive DNA have led to novel conclusions about repetitive sequence organization. Re-evaluation of the hyperchromicity techniques commonly used to determine the average length of genomic repetitive DNA regions indicates that both the extent of reassociation, and the possibility of non-random elution of hyperpolymers from hydroxyapatite can radically affect the observed hyperchromicity. An alternative interpretation of hyperchromicity experiments, presented here, suggests that the average length of repetitive regions in Syrian hamster DNA must be greater than 4000 nucleotides.S₁ nuclease digestion of reassociated 3200 nucleotide Syrian hamster repetitive DNA, on the other hand, yields both long (>2000 nucleotides) and short (300 nucleotides) resistant DNA duplexes. Calculations indicate that the observed mass of short nuclease-resistant duplexes (>60%) is too large to have arisen only from independent short repetitive DNA sequences alternating with non-repetitive regions. Reassociation experiments using long and short S₁ nuclease-resistant duplexes as driver DNA indicate that all repetitive sequences are present in both fractions at approximately the same concentration. Isolated long S₁ nuclease-resistant duplexes, after denaturation, renaturation, and a second S₁ nuclease digestion, again produce both long and short DNA duplexes. Reassociation experiments indicate that all repetitive DNA sequences are still present in the “recycled” long S₁ nuclease-resistant duplexes. These experiments imply that many of the short S₁ nuclease-resistant repetitive DNA duplex regions present in reassociated Syrian hamster DNA were initially present in the genome as part of longer repetitive sequence blocks. This conclusion suggests that the majority of “short” repetitive regions in Syrian hamster DNA are organized into scrambled tandem clusters rather than being individually interspersed with non-repetitive regions.     

A simplified method for mapping deletion/substitution mutants of bacteriophage lambda.

A convenient and rapid method for mapping deletion/substitution mutants of phage λ is presented. The method involves: (a) forming heteroduplex DNA molecules between the wild-type and the mutant: (b) digestion of the single-stranded regions with S₁ nuclease; (c) cleavage of a portion of the remaining duplex DNA with EcoRI nuclease or any convenient restriction endonuclease; and (d) separation of the resulting DNA fragments by gel electrophoresis. Using three deletion/substitution mutants with known endpoints, we show that the values obtained by this method deviate, on the average, by ±120 base pairs from the values obtained by electron microscopy.     

Evidence against the specific initiation of Okazaki fragments at the internucleosomal linkers

We have digested nuclei, isolated from [³H] thymidine pulse labelled cells, with nuclease S₁. Short pulse labelled DNA fragments were excised by the enzyme and released upon subsequent treatment with 2 M NaCl. Only a small fraction of the label was released from the S₁ digested nuclei by 0.5 M NaCl indicating that the cleavage sites were located in the DNA of the nucleosome cores. The results are not compatible with the hypothesis that the initiation of the Okazaki fragments occurs at the internucleosomal linkers.     

N-2-acetylaminofluorene modification of poly(dG-m5dC)·poly(dG-m5dC) induces the Z-DNA conformation

Poly(dG-m⁵dC)·poly(dG-m⁵dC) was modified by treatment with N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) and its conformation examined by circular dichroism (CD) and susceptibility to S₁ nuclease digestion. A sample with a modification level of 10% shows a CD spectrum characteristic of the Z form and is resistant to digestion by S₁ nuclease. The relative reactivity of several polymers with N-Aco-AAF was shown to follow the order of ease of formation of Z DNA: poly(dG-m⁵dC)·poly(dG-m⁵dC) > poly(dG-dC)·poly(dG-dC) > poly(dG)·poly(dC). This suggests that AAF reacts more readily with Z DNA than B DNA.     

Single-stranded regions in DNA from pre- and postmenopausal human ovaries.

No differences were noted between the percent (2–4%) of single-stranded regions in DNA isolated from pre- and postmenopausal human ovaries as determined by nuclease S₁ digestion.     

Uptake of homologous single-stranded fragments by superhelical DNA. III. The product and its enzymic conversion to a recombinant molecule

When superhelical DNA (RFI)² of phages φX174 or G4 takes up a homologous single-stranded fragment, RF DNA and fragment are linked by as many as 300 base-pairs, and a corresponding length of one strand of the RFI is displaced, forming a displacement loop (D-loop). The length of the base-paired region was estimated from the fraction of the associated ³²P-labeled fragment that was resistant to digestion by exonuclease VII, as well as by electron microscopy. Dissociation of the fragment by heating was characterized by a sharp melting curve. The displaced strand of the RF DNA was digested by two endonucleases that act on single-stranded DNA, the S₁ nuclease of Aspergillus oryzae and the recBC DNAase of Escherichia coli. Acting on complexes, both enzymes converted the form I [³H]DNA into form II DNA, and left some of the associated ³²P-labeled fragment undigested. The remaining ³²P-labeled fragment could no longer be displaced by branch migration, as expected if the displaced strand of the RF DNA were digested. The action of S₁ nuclease also produced the amount of acid-soluble ³H expected from digestion of the D-loop. Treatment of such digested complexes with polynucleotide ligase covalently linked about 35% of the remaining ³²P-labeled fragment to ³H-labeled strands, which proves that S₁ nuclease digested the D-loop.     

Alternative conformations of DNA modified by N-2-acetylaminofluorene

Modification of DNA by the carcinogen N-acetoxy-N-2-acetylaminofluorene gives two adducts, a major one at the C-8 position of guanine and a minor one at the N-2 position with differing conformations. Binding at the C-8 position results in a large distortion of the DNA helix referred to as the “base displacement model” with the carcinogen inserted into the DNA helix and the guanosine displaced to the outside. The result is increased susceptibility to nuclease S, digestion due to the presence of large, single-stranded regions in the modified DNA. In contrast, the N-2 adduct results in much less distortion of the helix and is less susceptible to nuclease S₁ digestion. A third and predominant adduct is formed in vivo, the deacetylated C-8 guanine adduct. The conformation of this adduct has been investigated using the dimer dApdG as a model for DNA. The attachment of aminofluorene (AF) residues introduced smaller changes in the circular dichroism (CD) spectra of dApdG than binding of acetylaminofluorene (AAF) residues. Similarly, binding of AF residues caused lower upfield shifts for the H-2 and H-8 protons of adenine than the AAF residues. These results suggest that AF residues are less stacked with neighboring bases than AAF and induce less distortion in conformation of the modified regions than AAF. An alternative conformation of AAF-modified deoxyguanosine has been suggested based on studies of poly(dG-dC)·poly(dG-dC). Modification of this copolymer with AAF to an extent of 28% showed a CD spectrum that had the characteristics of the left-handed Z conformation seen in unmodified poly-(dG-dC)·poly(dG-dC) at high ethanol or salt concentrations. Poly(dG)·poly(dC), which docs not undergo the B to Z transition at high ethanol concentrations, did not show this type of conformational change with high AAF modification. Differences in conformation were suggested by single-strand specific nuclease S₁ digestion and reactivity with anticytidine antibodies. Highly modified poly(dG-dC)·poly(dG-dC) was almost completely resistant to nuclease S₁ hydrolysis, while, modified DNA and poly(dG)·poly(dC) are highly susceptible to digestion. Two possible conformations for deoxyguanosine modified at the C-8 position by AAF are compared depending on whether its position is in alternating purine-pyrimidine sequences or random sequence DNA.     

A simple linear transform for the Folin-Lowry protein calibration curve to 1.0 mg/ml

The stability of covalently mercurated DNAs during DNA:DNA reassociation, heteroduplex recovery on sulfhydryl-Sepharose, and S₁ nuclease digestion under a variety of solvent and temperature conditions is described. The nonspecific loss of²⁰³Hg from mercurated DNA can be minimized by use of aqueous formamide solvents in reassociation experiments and by minimizing exposure to sulfhydryl reagents and temperatures above 35°C. Single-stranded DNA is shown to be more sensitive to demercuration than is native, duplex DNA.     

Investigation into the use of Aspergillus oryzae S1 nuclease in the presence of solvents which destabilize or prevent DNA secondary structure: Formaldehyde,formamide, and glyoxal

The activity of Aspergillus oryzae S₁ nuclease in solutions which destabilize DNA secondary structure was investigated. S₁ nuclease is able to degrade single-stranded DNA in the presence of various concentrations of formaldehyde, formamide, and glyoxal. It is further shown that S₁ nuclease can be used: (1) in the presence of formaldehyde to generate cleavage products from partially denatured duplex DNA; (2) to obtain thermal-melting profiles in the presence of formamide.     

Correlation of enzyme-induced cleavage sites on negatively superhelical DNA between prokaryotic topoisomerase I and S1 nuclease

Negatively superhelical pNS1 DNA with a molecular weight of 2.55 MDa (4 kbp) was found to contain 13 specific, unbasepaired sites that are sensitive to a single-strand-specific S₁ nuclease cleavage. The S₁-cleavage occurred once at these sites. In the absence of added Mg²⁺, the topoisomerase I purified from Haemophilus gallinarum formed a complex with the superhelical pNS1 DNA which has a hidden strand cleavage. Extensive proteinase K digestion of the complex led to cleavage of the DNA chain. Then the proteinase K-cleaved product was digested with S₁, which can cut the opposite strand at the preexisting strand cleavage to generate unit-length linear DNA. Restriction endonuclease analysis of the linear DNA shows that the topoisomerase-induced cleavage occurred once at ten specific sites on the DNA. The topoisomerase caused mainly single-strand cleavage at these sites, but infrequently also caused double-strand cleavage at the same sites. Of interest is the fact that these sites considerably coincide with the S₁-cleavable, unbasepaired sites.     

Nascent DNA in nucleosome like structures from chromatin

We have used chromatin sensitivity to cleavage by micrococcal nuclease as a probe for differences between chromatin containing nascent DNA and that containing bulk DNA. Micrococcal nuclease digested the nascent DNA in chromatin of swimming blastulae of sea urchins more rapidly to acid-soluble nucleotides than the DNA of bulk chromatin. A part of the nascent DNA occurred in micrococcal nuclease-resistant structures which were either different from, or temporary modifications of, the bulk nucleosomes. This was inferred from the size differences between bulk and nascent DNA fragments in 10% polyacrylamide gels after micrococcal nuclease digestion of nuclei from a mixture of ¹⁴C-thymidine long- and ³H-thymidine pulse-labeled embryos. Bulk monomer and dimer DNA fragments contained about 170 and 410 base pairs (bp), respectively, when 18% of the bulk DNA had been rendered acid-soluble. At this level of digestion, “nascent monomer DNA” fragments of about 150 bp as well as 305 bp “large nascent DNA fragments” were observed. Increasing levels of digestion indicated that the large nascent DNA fragment was derived from a chromatin structure which was more resistant to micrococcal nuclease cleavage than bulk dimer chromatin subunits. Peaks of ³H-thymidine-labeled DNA fragments from embryos which had been pulse-labeled and then chased or labeled for several minutes overlapped those of ¹⁴C-thymidine long-labeled monomer, dimer and trimer fragments. This indicated that the chromatin organization at or near the replication fork which had temporarily changed during replication had returned to the organization of its nonreplicating state.     

Copper-rich nucleoprotein generated by micrococcal nuclease

Nuclei from calf thymus tissue digested with micrococcal nuclease under nonchelating conditions yielded soluble nucleoprotein enriched in copper. Following limited digestion, the ratio of μg Cu:mg DNA was inversely related either to percent solubility of chromatin or to levels of enzyme maintaining an enzyme:A ₂₆₀ ratio of 0.059. The enzyme appeared to cleave preferentially regions of chromatin where copper is localized, releasing no additional metal upon further digestion. Moreover, the highest copper: DNA ratio was always associated with the least-digested sample. The distribution between copper and angiotensin II (AII) in chromatin fragments following slight nuclease digestion suggests a possible link between copper and nuclear AII binding. When nuclei are incubated with AII prior to digestion and dialysis, solubilized chromatin contained about three times more copper than buffer control. Metal profiles generated from gel (A-5 M) chromatography for these samples were distinctive: copper peaks appeared near or adjacent to linker DNA regions, and in the case of AII, coincided with fragments containing specific AII receptors; thus, there appears to be an enrichment of copper in these active nucleoprotein fragments.     

Interaction of quercetin with DNA

I n vitro experiments to study interaction of the mutagenic flavonoid quercetin with DNA are described. Calf thymus DNA treated with quercetin for various time periods was subjected to S₁ nuclease hydrolysis. Thermal melting profles of treated DNA were also determined using St nuclease. The rate of DNA hydrolyzed after 1 hr of pre-treatment with quercetin was found to be only about 50% of that in its absence. However, after 10 and 24hrs of treatment with the drug, the rate of S₁ nuclease hydrolysis was observed to be greater than that of native DNA. Thermal melting profiles of DNA, treated with quercetin for 10 and 24 hrs, indicated a slight decrease in melting temperatures. Gel filtration of native DNA, which had been digested with S₁ nuclease after preincubation with quercetin for 24 hrs, indicated the production of various sized degraded molecules. The results suggest that the initial interaction of quercetin with DNA may have a stabilizing effect on its secondary structure, but prolonged treatment leads to an extensive disruption of the double helix.     

Comparison of the subunit organization of early and late replicating chromatin

A comparison was made of the subunit organization of chromatin from regions of the genome with different metaphase chromosome banding characteristics by analyzing the accessibility of early and late replicating DNA in synchronized Chinese hamster ovary cells to digestion with staphylococcal nuclease. Three measures of nuclease susceptibility were employed: (1) the release of acid-soluble material; (2) a digestion index, P, which corresponds to the proportion of internucleosome segments which experienced at least one cleavage event; and (3) the size distribution of DNA fragments isolated from digested chromatin. Little or no difference was observed in the initial rates with which nuclease converted early and late replicating chromatin to acid-soluble material, although the initial digestion rates varied with time of cell collection in the cycle (metaphase > G1 mid-S > late-S or G2). Measurements of the digestion indices of material isolated from interphase cells suggested that initial cleavage events were more rapid in early replicating chromatin than in late replicating chromatin. In contrast, electrophoretic analysis revealed that oligomer DNA fragments from early labelled metaphase chromatin were slightly larger than corresponding fragments from late labelled metaphase chromatin. The size distribution of DNA in submonomer fragments obtained from extensively digested chromatin appeared to be identical regardless of the timing of replication or cell collection. Those small differences in chromatin digestibility that were observed may reflect subtle variations in the accessibility of internucleosome regions or perhaps in the higher-order arrangement of nucleosomes. However, no gross variation in accessibility to staphylococcal nuclease digestion was observed in chromatin localized to metaphase chromosome regions with vastly different cytological staining properties.     

Action of the S1 endonuclease from Aspergillus oryzae on simian virus 40 supercoiled component I DNA

The digestion products of superhelical component I of SV40 DNA incubated with various concentrations of nuclease S₁ from $\text{Aspergillus Oryzae}$, an enzyme specific for single-stranded nucleic acid, were studied. The enzyme shows a preference for supercoiled DNA I as opposed to relaxed DNA II molecules, and converts SV40 DNA I into linear molecules. Conditions have been developed under which the majority of SV40 DNA I molecules is converted into form II DNA. By using high concentrations of enzyme, it was possible to introduce further breaks in the DNA molecule; by increasing ionic strengh or using SDS this activity was not eliminated.     

DNA sequence organisation in avian genomes

By means of renaturation kinetics of DNA of the three avian species Cairina domestica, Gallus domesticus and Columba livia domestica the following major DNA repetition classes were observed: a very fast reannealing fraction comprising about 15% of the DNA, a fast or intermediate reannealing fraction that makes up 10%, and a slow reannealing fraction of about 70%, which apparently renatures with single copy properties. — Comparing the reassociation behaviour of short (0.3 kb) and long (>2 kb) DNA fragments of duck and chicken it becomes apparent that only 12% (duck) and 28% (chicken) of the single copy DNA are interspersed with repetitive elements on 2 to 3 kb long fragments. The lengths of the repetitive sequences were estimated by optical hyperchromicity measurements, by agarose A-50 chromatography of S₁ nuclease resistant duplexes and by electron microscopic measurements of the S₁ nuclease resistant duplexes. It was found that in the case of the chicken DNA the single copy sequences alternating with middle repetitive ones are at least 2.3 kb long; the interspersed moderate repeats have a length average of at least 1.5 kb. The sequence length of the moderate repeats in duck DNA is smaller. The results show that the duck and the chicken genomes do not follow the short period interspersion pattern of genome organisation, characteristic of the eucaryotic organisms studied so far.     

Changes in structural integrity of heart DNA from aging mice

The state of the structural integrity of the DNA from mouse myocardial cells has been investigated by utilizing both CsCl density gradient sedimentation and digestion by S₁ endonuclease from $\text{Aspergillus}$$\text{orzae}$. The DNA from myocardial cells of young mice sedimented in a narrow peak at the expected density of 1.701 g/cm³, while the DNA from the heart cells of senescent mice became broadly distributed in CsCl gradients, banding even more multimodally in alkaline sucrose gradients. This mode of sedimentation indicates that old mouse DNA becomes partially fragmented. When the native DNA of myocardial cells from 6, 20 and 30 month old mice was treated with single-strand specific S₁ endonuclease, it was the DNA from the senescent mice that showed a progressive increase in sensitivity to digestion by the enzyme. The results indicate that the heart DNA of aging mice develops single-stranded gaps in addition to a breakdown into differently sized fragments.