No abstract

The bis-benzimidazole drug Hoechst 33258 has been co-crystallized with the dodecanucleotide sequence d(CGCAAATTTGCG)2. The structure has been solved by molecular replacement and refined to an R factor of 18.5% for 2125 reflections collected on a Xentronics area detector. The drug is bound in the minor groove, at the five base-pair site 5'-ATTTG and is in a unique orientation. This is displaced by one base pair in the 5' direction compared to previously-determined structures of this drug with the sequence d(CGCGAATTCGCG)2. Reasons for this difference in behaviour are discussed in terms of several sequence-dependent structural features of the DNA, with particular reference to differences in propeller twist and minor-groove width.

The three‐dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti‐trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5′‐AAT‐3′ region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen‐bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen‐bonded contact with an amidinium nitrogen atom, N3 of the 5′ end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.

The FMN-dependent flavoprotein nitroreductase from Escherichia coli B (NTR) is used in cancer chemotherapy to activate a range of prodrugs. The crystal structure of this enzyme has been determined, using molecular replacement methods and refined at 2.06 A resolution. The recombinant 24-kDa enzyme was crystallized in the tetragonal space group P4(1)2(1)2, with unit cell dimensions of a = b = 57.74 A and c = 275.51 A and two molecules in the asymmetric unit. The structure has a final R factor of 20.3% (R(free) = 26.7%), for all data between the resolution ranges of 10-2.06 A, and includes 4453 protein atoms, 230 water molecules, and 2 flavin mononucleotide (FMN) molecules. The functional unit is a homodimer, which forms the asymmetric unit in the crystal structure. The tertiary structures of these two monomers and their subunit interactions are nearly identical. The molecular replacement search model, the crystal structure of the major NAD(P)H:FMN oxidoreductase of Vibrio fisheri (FRase 1), was selected on the basis of its high sequence identity to that of NTR. The final superposition of these two enzymes revealed a very similar overall fold, with variation in the structures focused around surface loops and helices near the FMN cofactor. Helix G is implicated in substrate specificity and is better resolved in the present NTR structure than in the previously reported FRase 1 structure. The FMN binding pocket is also well-resolved, showing the presence of two channels leading into the active site. The amino acid side chains and main chain atoms interacting with the FMN are well-ordered. The structure of the substrate binding pocket has been used to examine substrate specificity and enzyme kinetics for prodrugs used in antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT).

The structure of the G-G mispaired dodecanucleotide d(CGCGAATTGGCG)2 has been solved by x-ray crystallography and refined to an R factor of 18.8% at 2.2 A resolution for 3513 reflections. The dodecamer crystallizes as a B-type DNA double helix. It contains two G(and){G(syn) base pairs-i.e., G-4/G-16(antd)G-21/G-9(syn). The Hoogsteen base pairing involves atoms 0-6 and N-7 of the guanne in the syn conformation with atoms N-1 and N-2 of the anti-paired purine. One G-G base pair has a bifurcated hydrogen bond between G-4(N-1).G-21(N-7) and G-4(N-1)-G-21(O-6). There is little overall structural distortion of the double helix induced as a consequence of the mispiring. The helical width is significantly increased by comparison with the structure of the native duplex, and the minor groove width in the 5'-AATT region is decreased. The GIG base pairing induces high-BIu phosphate conformations at residues G-9 and T-20 in addition to more normal Be conformations at G-10 and G-22. It is suggested that these backbone aberrations provide signals for the facile repairability of GIG mispairs in DNA.DNA base mispairings or mismatches occur as a consequence of errors in genetic recombination and/or replication (1-3). These errors are corrected by a repair system that recognizes the lesion, unwinds the duplex, and excises the newly synthesized strand. Purine-containing mispairs occur more frequently than pyrimidine-pyrimidine pairs, with the ARC and G-G mispairings being the most efficiently repaired. G-G base pairing plays a major role in stabilizing telomere structure (4-9) and is important in purine-purine DNA triplexes (10) and in various RNA stem bulges (11).Several models for G-G-mispaired structures have been proposed (see, for example, refs. 6, 8, 12, and 13). A G(anti)-G(syn) base arrangement has been suggested for the GIG pairing in a DNA duplex, while NMR data and molecular models suggest that the guanines are hydrogen-bonded in a cyclic fashion for the four-stranded quartet structure in telomeric DNA. NMR solution data for the d(GAGGAGGACG)-d(CGTGCGTCCTC) duplex, containing a central G-G mispair (14), suggest that the bases adopt a symmetrical anti-syn arrangement and are stacked into the helical axis. The hydrogen-bonding pattern preferred by these authors involves symmetric hydrogen bonding between the N-1 imino protons and the 0-6 carbonyl oxygen atom. A recent solution NMR analysis of the d(CGCGAATTGGCG)2 duplex indicates that the G(anti)-G(anti) conformation is favored at low temperatures and under high-salt conditions, with the structure characterized by non-hydrogen-bonded G&G base pairs. This conformation is, however, unstable at higher temperatures, as judged by spectral heterogeneity, with the mispairing bases providing a focus for localized melting. Localized DNA bulges were considered to result from unusual phosphate backbone torsion angles in the vicinity of the G-G sites (15). In contrast, the recently reported crystal structure and NMR analysis of the four-stranded d(G4T4G4)2 sequence from the 3'...