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Inovirus C2 is 1295 nm long and 6.8 nm in diameter, and its mass is 24 million Da. Its genome is a topologically circular, single-stranded DNA molecule of 8100 nucleotides. The DNA is packed in the virion as two antiparallel strands, with a rise per nucleotide in each strand of 3.2 A; it can be assigned spectroscopic properties like those of base-stacked, right-handed, double-stranded DNA. The stoichiometric ratio (n/s) of nucleotides to subunits of the major coat protein is close to 2. The protein subunit contains 52 amino acids, and the DNA sequence of its gene does not encode a signal peptide. The protein conformation in the virion is helical, mostly alpha-helix with perhaps some 3(10)-helix. The amino acid sequence of the DNA interaction domain of the subunit is unique among Inovirus species. On the basis of its coat protein sequence and available theories of helical symmetry in such structures, C2 appears to be either an unusual member of filamentous virus symmetry class II or the defining member of a new symmetry class.

It is widely accepted that mutagenic DNA lesions fall into two categories: mispairing lesions hydrogen bond with an incorrect incoming base, generally do not stop replication, and possess high mutagenic efficiency without any requirement for induced functions; noninstructional lesions lack accessible template information, act as strong blocks to DNA replication (and are therefore toxic), and their mutagenic effects are SOS-dependent. Our recent results show that ethenocytosine (epsilon C), a noninstructional exocyclic DNA lesion induced by vinyl chloride, may have unusual mutagenic properties. To obtain more definitive experimental evidence for the observed effects, we have introduced a single epsilon C residue at a specific site of coliphage M13AB28 replicative form DNA by a "single-stranded linker-ligation" technique. The resulting DNA was purified and transfected into appropriate recA+ or recA- Escherichia coli host cells. The effect of epsilon C on survival was determined from transfection efficiency. Both the frequency and specificity of mutations induced by epsilon C were determined by direct sequence analysis of randomly picked progeny phage plaques. The results indicated that epsilon C has little effect on the survival of M13 DNA. Approximately 30% of the progeny phage obtained by transfecting epsilon C DNA had a base substitution mutation precisely at the lesion site. No such mutations were observed in progeny plaques obtained by transfecting the control DNA construct. All epsilon C-induced mutations were either C-to-T transitions or C-to-A transversions. Neither survival nor mutagenic efficiency was significantly affected in recA- host cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The single-stranded packaged genome (ssDNA) of bacteriophage phi X174 is shown by Raman spectroscopy to lack both the ordered phosphodiester backbone and base stacking, which are demonstrated for unpackaged, protein-free ssDNA. In solutions of moderate ionic strength, unpackaged ssDNA contains 36 +/- 7% of deoxyribosyl phosphate groups with conventional B-type backbone geometry [i.e., gauche- and trans orientations, respectively, for the 5'O-P (alpha) and 3'O-P (zeta) torsions], indicative of hairpin formation and intramolecular base pairing. Additionally, the bases of unpackaged ssDNA are extensively stacked. Estimates from Raman band hypochromic effects indicate that unpackaged ssDNA contains approximately 70% of the maximal base stacking exhibited in the linear, double-stranded, replicative form III of phi X174 DNA. Conversely, for the packaged phi X174 genome, ordered (B-type) phosphodiester groups are not present, and only 40% of the base stacking in RFIII DNA is observed. These results are interpreted as evidence that the substantial hairpin-forming potential of ssDNA is eliminated by specific and extensive ssDNA-protein interactions within the phi X174 virion. Comparison of the present results with studies of other packaged single-stranded nucleic acids suggests that proteins of the capsid shell (gpF + gpG + gpH) do not fully account for the conformational constraints imposed on ssDNA of phi X174. Accordingly, we propose a model for ssDNA packaging in which the small basic gpJ protein, which is packaged along with the genome, is involved stoichiometrically in binding to the ssDNA (approximately 90 nucleotides per subunit). The proposed gpJ-DNA interactions could prevent helical hairpin formation, restrict base stacking, and disfavor fortuitous base pairing within the capsid. The present analysis is based upon use of model nucleic acids of known conformation for calibration of the Raman intensity in the region 810-860 cm-1 in terms of specific secondary structures. The calibration curve allows quantitative determination of the percentage of ssDNA nucleotides for which the 5'O-P-O3' group is configured (g-,t) as in the B-form of DNA. The method proposed here is analogous to that employed by Thomas and Hartman (1973) for ssRNA and should be applicable to single-stranded DNA and to partially denatured forms of double- and multiple-stranded DNAs.

The method of alkaline elution provides a sensitive measure of DNA single-strand length distribution in mamalian cells and is applicable to a variety of problems concerning DNA damage, repair, and replication. The physical basis of the elution process was studied. The kinetics of elution above the alkaline transition pH were found to occur in two phases: an initial phase in which single-strand length is rate limiting, followed by a phase in which elution is accelerated due to the accumulation of alkali-induced strand breaks. The range of DNA single-strand lengths that can be discriminated by elution above the alkaline transition pH was estimated by calibration relative to the effects of x ray, and was found to be 5 X 10(8)-10(10) daltons. Shorter DNA strands elute within the pH transition zone, which extended from pH 11.3 to 11.7 when tetrapropylammonium hydroxide was used as base. This elution was relatively rapid, but was sharply limited by pH, according to the length of the strands: the length of the strands eluted increased with increasing pH. Alkaline elution was inhibited by treatment of cells with low concentrations of nitrogen mustard, a bifunctional alkylating known to cross-link DNA. On investigation of the possibility that DNA subclasses may differ in their elution behavior, satellite L strands were found to elute more slowly from cells exposed to a low dose of x ray than did the bulk DNA.

Ionic strength studies using homogeneous preparations of chicken erythrocyte nucleosomes containing either 146 or 175 base pairs of DNA show a single unfolding transition at about 1.5 mM ionic strength as determined by small-angle neutron scattering. The transition seen by some investigators at between 2.9 and 7.5 mM ionic strength is not observed by small-angle neutron scattering in either type of nucleosome particle. The two contrasts measured (H2O and D2O) indicate that only small conformational changes occur in the protein core, but the DNA is partially unfolded below the transition point. Patterson inversion of the data and analysis of models indicate that the DNA in both types of particle is unwinding from the ends, leaving about one turn of supercoiled DNA bound to the histone core in approximately its normal (compact) conformation. The mechanism of unfolding appears to be similar for both types of particles and in both cases occurs at the same ionic strength. The unfolding observed for nucleosomes in this study is in definite disagreement with extended superhelical models for the DNA and also disagrees with models incorporating an unfolded histone core.

Secondary cultures of normal rat embryo cells were synchronized by a double thymidine block and pulsed with 10(-7) M 5-[3H]bromodeoxyuridine (BrdUrd) OR 10(-7) M[3H]thymidine during an entire S phase (7.5 h). To examine the pattern of [3H]thymidine, DNA was immediately extracted and purified at the completion of the S phase, CsCl density gradient centrifugation revealed that substitution for thymine by bromouracil was less than 7%. Single-strand specific nucleases obtained from Aspergillus oryzae and Neurospora crassa were allowed to react with native and partially depurinated (24-29%) [3H]BrdUrd-labeled rat DNA samples, and the products were assayed by hydroxylapatite column chromatography. Approximately 4-6% of the native, nondepurinated rat DNA was hydrolyzed by both nucleases. However, 24-28% of the partially depurinated, [3H] thymidine-labeled rat DNA was hydrolyzed by both enzymes as determined by loss of mass as well as radioactivity. Whereas comparable levels of depurinated, [3H]BrdUrd-labeled DNA were physically hydrolyzed by both nucleases, nearly 65% of the radioactivity was not recovered. Native, as well as depurinated, enzyme-treated DNA samples were sequentially and preparatively reassociated into highly repetitive, middle repetitive, and nonrepetitive nucleotide sequence components. The absolute and relative specific activities of each subfraction of native [3H]thymidine-labeled DNA were comparable. [3H]BrdUrd was differentially concentrated in the middle repetitive sequences as compared to other reiteration frequency types. When depurinated, nuclease-treated DNA samples were similarly fractionated, [3H]thymine moieties were uniformly distributed thoughout all sequences. However, a differential loss of [3H]BrdUrd moieties was detected predominantly from the middle repetitive nucleotide fraction. Melting profiles of the renatured DNA samples were characteristic of each respective DNA subfraction regardless of isotopic precursor. These results suggest that [3H]BrdUrd may be differentially incorporated into A + T rich clusters of rat DNA, especially in the moderately repeated chromosomal elements.

We have investigated structural change in rat liver DNA produced by different isolation procedures and specifically compared the integrity of DNA derived by phenol extraction from isolated and purified nuclei with preparations extracted immediately from a crude liver homogenate containing intact nuclei. As indicated by stepwise elution from benzoylated DEAE-cellulose, most structural change in DNA was evident following nuclei isolation. Damage principally involved generation of single-stranded regions in otherwise double-stranded DNA fragments; totally single-stranded DNA was not detected by hydroxylapatite chromatography. Caffeine gradient elution suggested formation of single-stranded regions extending for up to several kilobases. In neutral sucrose gradients, differences in sedimentation rates of respective DNA samples consequent upon S1 nuclease digestion could be detected after isolation of nuclei, though not in other circumstances. The observed single-strand-specific nuclease digestion of DNA could apparently be reduced if steps were taken to reduce autodigestion during nuclei isolation by reduction of temperature and covalent cation concentration. The results are discussed in terms of the use of exogenous and endogenous nucleases in chromatin fractionation studies involving isolated nuclei and possible artifactual findings that may be generated by single-strand-specific autodigestion.

We have looked for the presence of single-stranded DNA in human melanoma cells. Single-stranded DNA was observed by lysis of cells in dilute alkali (to partly denature the DNA) followed by CsCl gradient centrifugations. In normally growing cells we did not observe single-stranded DNA whereas large amounts were present in cells treated with aphidicolin (an inhibitor of DNA polymerase alpha). The single-stranded DNA is much larger (greater than 20 kb) than Okazaki fragments. When the cells were washed free of aphidicolin, the single-stranded DNA was converted to high molecular weight DNA. Furthermore, when DNA synthesis is recovering after drug treatment, the single-stranded DNA disappears. The single-stranded DNA represents a transient step during the maturation of newly synthesized DNA.

Initial results of an approach to the isolation of functionally active chromatin are described. Slight digestion of mouse myeloma nuclei at 0 degrees C with micrococcal nuclease, followed by dialysis against near-physiological saline solution containing 1 mM Mg2+, caused release of up to 17% of the nuclear DNA as soluble nucleoproteins. This soluble (S) fraction was relatively depleted in H1 histones and methylated DNA (5-methylcytosine) but highly enriched in RNA, single-stranded DNA, and nonhistone chromosomal proteins, particularly two species of the high mobility group identified as HMG 1 and HMG 2. The S fraction released most rapidly (6--8% of the total DNA) consisted mainly of mono- and small oligonucleosomes. The mononucleosomes appeared normal in terms of sedimentation behavior, DNA length, and content of histones H2A, H2B, H3, and H4, but lacked H1, and instead were associated with approximately stoichiometric amounts of HMG 1 and HMG 2. Studies using isolated, fluorescence-labeled, total mouse HMG proteins indicated that added HMG 1 and HMG 2 do not bind strongly to S-fraction nucleoproteins but that two smaller HMG species (probably HMG 14 and HMG 17) do bind preferentially to S-fraction mono- and dinucleosomes. These results argue against artifactual redistribution of HMG 1 and HMG 2 during this fractionation but suggest caution in interpreting the distribution of smaller HMG proteins after digestion of chromatin. The potential relationship of this soluble fraction to transcriptionally active chromatin is discussed.

The electrophoresis of various DNA restriction fragments ranging in size from 47 to 6000 base pairs has been examined as a function of agarose concentration, electric field strength, and time of electrophoresis. A typical sigmoidal curve was obtained when the logarithm of the molecular weight was plotted as a function of mobility. The logarithms of the mobilities of all fragments were a linear function of gel concentration, if the mobilities of fragments greater than or equal to 1000 base pairs were first extrapolated to zero electric field strength. The slopes of the lines, called the retardation coefficients, were found to be linearly proportional to the effective hydrodynamic surface areas of the fragments, as predicted by the Ogston theory of pore size distribution. The logarithm of the mobility of native DNA fragments was inversely proportional to Mr0.8 over the entire molecular weight range, if the mobilities of fragments larger than 1000 base pairs were first extrapolated to zero electric field strength. The logarithm of the mobility of denatured, single-stranded DNA molecules was inversely proportional to the square root of molecular weight. The agreement of these results with the Ogston theory argues against a reptation mechanism for the movement of DNA molecules less than or equal to 6000 base pairs through agarose gels.