Isolation and characterization of self-complementary sequences from øX174 viral DNA

Bartok, Katalina; Harbers, Barbara; Denhardt, David T.

doi:10.1016/s0022-2836(75)80161-6

Cited by 13 publications

(1 citation statement)

References 42 publications

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“…Second, Edlind and Ihler (1980) compared longrange base pairing in linear and circular 0X174 ssDNA and found that the number, size, and location of loops were essentially the same for the two forms, although the frequency of small loop formation may have been higher in linear molecules. Third, a direct comparison of the resistance of the two phage DNAs to single-strand-specific nucleases (as a measure of double-strand formation) reveals that both 0X174 and fd circular forms contain only 2-3% of their bases in such structures (Bartók et al, 1975;Schaller et al, 1969). By this criterion the two DNAs appear to have similar secondary structures throughout their entire sequences.…”

Section: Discussionmentioning

confidence: 98%

Differences in secondary structure between packaged and unpackaged single-stranded DNA of bacteriophage .vphi.X174 determined by Raman spectroscopy: a model for .vphi.X174 DNA packaging

Benevides

Stow²,

Ilag³

et al. 1991

Biochemistry

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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.

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Section: Discussionmentioning

confidence: 98%