STUDIES OF VIRUS STRUCTURE BY LASER RAMAN-SPECTROSCOPY .38. SECONDARY STRUCTURE AND INTERACTIONS OF THE PACKAGED DSDNA GENOME OF BACTERIOPHAGE-P22 INVESTIGATED BY RAMAN DIFFERENCE SPECTROSCOPY

Vibrational spectra of the double-stranded DNA genome of bacteriophage P22 in packaged and unpackaged states are compared by digital difference Raman spectroscopy. The difference Raman spectrum, which is sensitive to structural changes at the level of <2% of a given nucleotide type, reveals the effects of packaging upon sugar pucker, glycosyl orientation, phosphodiester geometry, base pairing, base stacking, and the electrostatic environment of DNA phosphate groups. For both packaged and unpackaged states, the experiments were performed on aqueous solutions at 25-degrees-C containing effective P22 DNA concentrations of 30-50 mg/mL in 200 mM NaCl + 10 mM MgCl2 + 10 mM Tris at pH 7.5. At the experimental conditions employed, the B-form secondary structure of unpackaged P22 DNA is minimally perturbed by packaging the viral genome in the virion capsid. However,the electrostatic environment of DNA phosphates is dramatically altered with packaging. Specifically, we find the following: (1) C2'-endo sugar pucker and anti glycosyl orientations are conserved for all nucleosides. (2) Watson-Crick base pairing is essentially completely retained. (3) Alternative secondary structures, whether right- (A or C form) or left-handed (Z form), are not evident in either the packaged or unpackaged viral genome. (4) Small Raman hyperchromic effects (<10%) observed for certain marker bands of dG, dA, and dT in the packaged state of P22 DNA suggest slightly reduced base-stacking interactions with packaging. These are consistent with previously reported UV hyperchromic effects, but the Raman spectrum shows that they are not associated with either base unpairing or strand separation. (5) Marginal intensity changes within the envelope of the phosphodiester B-form Raman marker band (825-845 cm-1) are consistent with a small net change in DNA groove dimensions attendant with packaging, interpreted as a net narrowing of the minor groove of B-form DNA, as has been observed for AT-rich sequences. (6) A large intensity decrease in the phosphodioxy Raman marker band with packaging is shown by analogy with model compounds to reflect increased electrostatic shielding of phosphates for the packaged genome. On the basis of model compound studies, the observed effect is judged to be equivalent to an approximate 10-fold increase in the local Mg2+ concentration in the packaged state of P22 DNA. (7) We find no evidence in the Raman spectrum of specific intermolecular interactions involving capsid protein and major-groove sites of packaged DNA. These findings apply both to wild-type P22 and to a mutant variant in which dsDNA of approximately 5% greater mass than wild type has been packaged. The present results, which appear to disfavor extensive folding, kinking, or sharp bending of the packaged P22 DNA, are discussed in relation to proposed models for organization of condensed viral chromosomes of dsDNA bacteriophages.