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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 crystal structure of the decanucleotide d(CGCAATTGCG)2 has been solved by a combination of molecular replacement and heavy-atom procedures and has been refined to an R factor of 20.2% at 2.7 A. It is not a fully base-paired duplex but has a central core of eight WatsonCrick base pairs flanked by unpaired terminal guanosines and cytosines. These participate in hydrogen-bonding arrangements with adjacent decamer duplexes in the crystal lattice. The unpaired guanosines are bound in the G+C regions of duplex minor grooves. The cytosines have relatively high mobility, even though they are constrained to be in one region where they are involved in base-paired triplets with G-C base pairs. The 5'-AATT sequence in the duplex region has a narrow minor groove, providing further confirmation of the sequence-dependent nature of groove width.The crystal structures of many B-DNA oligonucleotide duplexes have now been determined (1-3). These can be divided into two principal classes: (i) duplexes (the overwhelming majority) with Watson-Crick hydrogen-bonding arrangements for G-C and A-T base pairs and (ii) duplexes with base-pair mismatches such as A-G, G-G, and G-T. Such mismatches are invariably accommodated within double-helical structures (4,5). Fully base-paired helices have also been observed to be the norm in protein-DNA complexes, even when the DNA is highly perturbed from a standard B form. The sole exceptions reported to date are (i) DNA structures with a looped-out region by virtue of insertion of nonpairing bases (6, 7) and (ii) the structure of the Hha I methyltransferase-d(TGATAGfl5C-GCTATC), where the 5-fluorocytosine base is swung out of the DNA helix into the enzyme active site (8). A Z-DNA hexamer crystal structure has been reported (9) in which the first cytosine of each duplex is swung out, forming a WatsonCrick base pair with a guanine in a symmetry-related duplex.The first crystal structure of a B-DNA oligonucleotide to be determined (10) was that of the self-complementary dodecanucleotide d(CGCGAATTCGCG)2. This showed features of sequence-dependent structure such as a narrow minor groove in the 5'-AATT region and high propeller twist for the A-T base pairs. These have been observed in other dodecanucleotide crystal structures (11), such as that of d(CGTGAATT-CACG)2 (12, 13), and are unlikely to be due to the influence of crystal packing forces. The crystal structures of both of the decamer duplex sequences d(CCATTAATGG)2 (14) and d(C-GATTAATCG)2 (15) show narrow minor grooves and high propeller twists for most of the A-T base pairs; however, the alternating sequence d(CGATATATCG)2 (16) does not have these features. We have determined the crystal structure of the self-complementary decamer d(CGCAATTGCG)2, which has close sequence analogy to the original dodecanucleotide structure (10). This provides an independent structural assessment of the sequence-dependent features of short runs of A-T base pairs. The decamer has B-DNA duplex geometry for its central eight base pairs, but with an unpreced...

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CRYSTALLOGRAPHY OF BIOLOGICAL MACROMOLECULES the orphaned 3U-tem1inal end in the minor groove of the adjacent duplex where it synm1etrically pairs with the 5'-terminal guanine to form a d[G*(G.C)] base-tliplet. Our findings extend to the minor groove a DNA hydrogen bonding pattem which permits basepair recognition dming homologous recombination.