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Previous studies have shown that a cationic water-soluble pyridinium zinc phthalocyanine (PPC) is a powerful photosensitizer that is able to inactivate Escherichia coli. In the current work incubation of E. coli cells with PPC in the dark caused alterations in the outer membrane permeability barrier of the cells, rendering the bacteria much more sensitive to hydrophobic compounds, with little effect seen with hydrophilic compounds. Addition of Mg 2؉ to the medium prior to incubation of the cells with PPC prevented these alterations in the outer membrane permeability barrier. The presence of Mg 2؉ in the medium also prevented the photoinactivation of E. coli cells with PPC. These results are consistent with the hypothesis that PPC gains access across the outer membrane of E. coli cells via the self-promoted uptake pathway, a mechanism of uptake postulated for the uptake of other cationic compounds across the outer membranes of gram-negative bacteria.

To investigate the nature of the chemical determinants in DNA required for nonspecific binding and bending by proteins we have created a novel DNA in which inosine-5-methylcytosine and 2,6-diaminopurine-uracil base pairs are substituted for normal base pairs in a defined DNA sequence. This procedure completely switches the patterns of the base pair H bonding and attachment of exocyclic groups. We show that this DNA binds a histone octamer more tightly than normal DNA but, surprisingly, does not alter the orientation of the sequence on the surface of the protein. However, in general, the addition or removal of DNA exocyclic groups reduces or increases, respectively, the affinity for the histone octamer. The average incremental change in binding energy for a single exocyclic group is Ϸ40 J͞mol. The orientation of the DNA in core nucleosomes also is sensitive to the number and nature of the exocyclic groups present. Notably, substitutionwiththenaturallyoccurringcytosineanalogue,5-methylcytosine, shifts the preferred rotational position by 3 bp, whereas incorporating 2,6-diaminopurine shifts it 2 bp in the opposite direction. These manipulations potentially would alter the accessibility of a protein recognition sequence on the surface of the histone octamer. We propose that exocyclic groups impose steric constraints on protein-induced DNA wrapping and are also important in determining the orientation of DNA on a protein surface. In addition, we consider the implications of the selection of A-T and G-C base pairs in natural DNA.The local deformation of DNA by DNA-bending proteins can substantially exceed the normal conformational fluctuations of DNA free in solution. Although the interactions that determine the local sequence-dependent conformation of free DNA are relatively well understood (1-7), the nature of the chemical constraints that limit its further deformation have not been explored extensively. The nucleosome core particle in which the central 125 bp of DNA are wrapped in 1.6 superhelical turns about the histone octamer (8, 9) provides a good system to study this problem. In this particle both the major and minor grooves are compressed on the inside of the wrapped DNA and widened on the outside. A major determinant of the rotational placement of the DNA in the nucleosome is the periodic occurrence of short A͞T-rich sequences in helical phase and of short G͞C-rich sequences in the opposite phase. These short sequences favor local conformations with narrow and wide minor grooves, respectively, and thus together facilitate the tight wrapping of DNA (10-16).To investigate the nature of other chemical determinants that constrain the wrapping of DNA around the histone octamer we chose a prokaryotic DNA sequence derived from the Escherichia coli tyrT promoter. Previous studies have shown that the histone octamer occupies a preferred rotational position on this DNA (10). We modified this DNA by replacing the naturally occurring bases with analogues either lacking or containing additional exocyclic groups b...

Mitomycin C (MC) is a G·C-specific antitumor antibiotic that alkylates and cross-links DNA at 2-amino groups of guanine residues. Both reactions are known to be enhanced at guanines in the CpG sequence by independent mechanisms. The mechanisms were probed by substituting 2,6-diaminopurine (D) into oligonucleotides and into a 162-bp tyrT DNA restriction fragment and determining their alkylation and cross-linking by MC. Covalent D-MC adducts were isolated and structurally characterized. The results indicated that 2,6-diaminopurine functioned as a substrate analogue of guanine and displayed enhanced reactivity toward MC in all systems. The observed TpD sequence selectivity of the modifications by MC was analogous to the CpG sequence selectivity of guanine modifications. Selective monoalkylation and cross-linking was observed also at the TpG·CpD sequence, indicating that two purine 2-amino groups are necessary and sufficient for the selectivity regardless of whether they are supplied by G or by D. These findings reinforce the previously proposed mechanism in which the selectivity of monoalkylation by MC is attributed to a specific H-bond between the drug and the 2-amino group of a guanine. The specific sequence required for D−D and D−G cross-link formation was established as TpD·TpD and TpG·CpD, respectively, determined by the same minor groove structural factors as in the CpG·CpG cross-linking process. MC cross-linking was also probed in a 162-bp tyrT DNA fragment in which Gs and As were replaced by inosine and 2,6-diaminopurine, respectively, using PCR. Cross-linked sites were quantitated and mapped on the basis of an empirical correlation between the electrophoretic mobility of cross-linked DNA and the position of the cross-links relative to the center of the sequence. In the natural DNA sequence hotspots for formation of MC-cross-links were identified. The cross-links were shown to be translocated from CpG·CpG in the natural DNA to TpD·TpD or TpG·CpD in the substituted DNAs, demonstrating the dominant role of the purine 2-amino group for cross-link site selection by MC.