X-ray analysis of the complex of netropsin with the B-DNA dodecamer of sequence C-G-C-G-A-A-T-TBrC-G-C-G reveals that the antitumor antibiotic binds within the minor groove by displacing the water molecules of the spine of hydration. Netropsin amide NH furnish hydrogen bonds to bridge DNA adenine N-3 and thymine 0-2 atoms occurring on adjacent base pairs and opposite helix strands, exactly as with the spine of hydration. The narrowness of the groove forces the netropsin molecule to sit symmetrically in the center, with its two pyrrole rings slightly non-coplanar so that each ring is parallel to the walls of its respective region of the groove. Drug binding neither unwinds nor elongates the double helix, but it does force open the minor groove by 0.5-2.0 A, and it bends back the helix axis by 8°across the region of attachment. The netropsin molecule has an intrinsic twist that favors insertion into the minor groove of B-DNA, and it is given a small additional twist upon binding. The base specificity that makes netropsin bind preferentially to runs of four or more ACT base pairs is provided not by hydrogen bonding but by close van der Waals contacts between adenine C-2 hydrogens and CH groups on the pyrrole rings of the drug molecule. Substitution of one or more pyrroles by imidazole could permit recognition of G'C base pairs as well, and it could lead to a class of synthetic "lexitropsins," capable of reading any desired short sequence of DNA base pairs.Netropsin and its close relative distamycin ( Fig. 1) are antiviral antitumor antibiotics that, although too toxic for clinical use, have received extensive study as the paradigms of base-specific yet non-intercalative DNA-binding drug molecules. First isolated from Streptomyces netropsis in 1951 (1, 2), netropsin exerts its biological activity by binding tightly to double-helical B-DNA, interfering with both replication and transcription (3, 4). It shows little or no affinity for single-stranded DNA or RNA or for double-stranded RNA or DNA-RNA hybrids (3-5), suggesting that it does not bind to the A helix. It also fails to bind to left-handed Z-DNA; in fact, binding of netropsin to DNA favors A-to-B and Z-to-B helix transitions (6, 7).Chemical protection studies (3,4,8) and Overhauser NMR experiments (9) indicate that netropsin does not intercalate between base pairs, but it binds within the minor groove of the intact double helix, using hydrogen bonds between netropsin amide NH and exposed adenine N-3 and thymine 0-2 on the floor of the minor groove. The drug molecule attaches to clusters of four or more A'T or ITC, but not to G-C, base pairs (3, 10, 11). Alternating A-T-A-T regions bind netropsin less well than continuous runs of A or T (12, 13). Binding involves both an electrostatic component from the two cationic ends and hydrogen bonds from the central three amide NH groups, although neither aspect is absolute- FIG. 1. The netropsin molecule can be regarded as being assembled from (left to right): guanidinium, amide, methylpyrrole, amide, methylpyrr...

No abstract

The crystal structure of the synthetic DNA dodecamer CGCATATATGCG has been solved at 2.2-A resolution.

The self-complementing dodecamer 5'-CGCGAATTCGCG-3' and its complexes with the antibiotic netropsin and the restriction endonuclease EcoRI provide substrates of known three-dimensional structure to study the stereochemistry and mechanism of the artificial nuclease of 1,10-phenanthroline-copper ion [(OP)2Cu+]. Analysis of the reaction products with the 5'-32P dodecamer on 20% sequencing gels has demonstrated the presence of 3'-phosphoglycolate ends in addition to 3'-phosphomonoester ends expected from previous studies. A reaction intermediate, which is a precursor to 3'-phosphomonoester termini, has been trapped; in contrast, no comparable species for the 5'-phosphomonoester termini can be detected when 3'-labeled DNAs are utilized as substrates. The reactive oxidative species formed by the coreactants (OP)2Cu+ and hydrogen peroxide is distinguishable in its chemistry from the hydroxyl radicals produced by cobalt-60 gamma-irradiation. The freely diffusible hydroxyl radicals generated by cobalt-60 irradiation produce equivalent amounts of 3'-phosphomonoester and 3'-phosphoglycolate termini whereas the 3'-phosphomonoesters are the preferred product of (OP)2Cu+ and H2O2. On the basis of the structures of the products obtained, the principal site of attack of the coordination complex is on the C-1 of the deoxyribose within the minor groove. This conclusion is supported by the footprinting of netropsin binding to the dodecamer. Crystallographic results have demonstrated that netropsin binds to the minor groove at the central AATT residue. A clear protection of attack by the coordination complex at the deoxyriboses associated with A-5, T-6, T-7, and C-9 is fully consistent with attack from the minor groove without intercalation during the course of the cleavage reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract