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N6-methyldeoxyadenosine (6mA) is a well-characterized DNA modification in prokaryotes but reports on its presence and function in mammals have been controversial. To address this issue, we established the capacity of 6mA-Crosslinking-Exonuclease-sequencing (6mACE-seq) to detect genome-wide 6mA at single-nucleotide-resolution, demonstrating this by accurately mapping 6mA in synthesized DNA and bacterial genomes. Using 6mACE-seq, we generated a human-genome-wide 6mA map that accurately reproduced known 6mA enrichment at active retrotransposons and revealed mitochondrial chromosome-wide 6mA clusters asymmetrically enriched on the heavy-strand. We identified a novel putative 6mA-binding protein in single-stranded DNA-binding protein 1 (SSBP1), a mitochondrial DNA (mtDNA) replication factor known to coat the heavy-strand, linking 6mA with the regulation of mtDNA replication. Finally, we characterized AlkB homologue 1 (ALKBH1) as a mitochondrial protein with 6mA demethylase activity and showed that its loss decreases mitochondrial oxidative phosphorylation. Our results show that 6mA clusters play a previously unappreciated role in regulating human mitochondrial function, despite 6mA being an uncommon DNA modification in the human genome.

Four [12]aneN3 modified tetraphenylethene (TPE) compounds with different numbers of polyamine units and structure configurations, namely 1, 2, 3, and 4, were designed and synthesized. All compounds showed strong aggregation-induced emission (AIE) features. Compounds 2 and 4 showed significant emission enhancement after the addition of ssDNAs and dsDNAs of different lengths as well as calf thymus DNA (ctDNA). Compounds 1 and 3 showed very poor fluorescent responses toward DNAs. Gel electrophoresis demonstrated the abilities of 1-4 to condense DNA effectively. Complete retardation of plasmid DNA can be achieved at a concentration of 25 μM (1), 8 μM (for 2 and 3) and 4 μM (4). Experiments including fluorescent contrastive titrations, scanning electron microscopy, dynamic laser scattering, EB displacement, and gel electrophoresis demonstrated that the four compounds were able to integrate with DNA through electrostatic interactions and supramolecular stacking. A vicinal configuration around TPE (2) and more triazole-[12]aneN3 recognition sites (4) evidently enhanced the sensing capability toward oligonucleotides, and the TPE unit played an important role in the plasmid DNA condensation process because of its strong binding. With the advantages of low cytotoxicity, effective DNA sensing, and DNA condensing properties, compound 4 was successfully applied as a nonviral DNA vector and fluorescent tracer for label-free gene delivery, which is the first example of a nonviral gene vector with AIE activity.

Two DNA cleavage agents, meso-dihydroguaiaretic acid (1) and isobavachalcone (2) together with the known alpha-ylangene, beta-sitosterol, daucosterol, pentacosane, hexacosanic acid and cerotic acid 1-monoglyceride were isolated from the stem barks of Kadsura ananosma Kerr for the first time. Compounds 1 and 2 showed relaxation of supercoiled DNA to nicked DNA. 1 represented a new structural type of DNA cleavage agent, while 2 was reported to show DNA strand-scission activity for the first time. 1 also showed significant cytotoxic effect on Hela and Leukemia cells in vitro.

The lignans, gomisin G (1), schisantherin A (2), benzoylgomisin Q (3) and isoanwulignan (4) were isolated from the stems of Schisandra henryi. Compound 1 showed moderate DNA strand scission activity and significant cytotoxic effect on leukemia and Hela cells in vitro. Compound 1 represents a new type of DNA strand scission agent.

The anticancer antibiotic chromomycin A3 (Chro) is a DNA minor groove binding drug belonging to the aureolic family. Chro likely exerts its activity by interfering with replication and transcription. Chro forms a dimer, mediated by a divalent metal ion, which binds to G/C-rich DNA. Herein we report the first crystal structure of Chro bound to d(TTG GCCAA)2 DNA duplex solved by multiwavelength anomalous diffraction (MAD) based on the chelated Co3+ ion. The structure of the Mg2+ complex was subsequently refined at 2.15 A resolution, which revealed two complexes of metal-coordinated dimers of Chro bound to the octamer DNA duplex in the asymmetric unit. The metal ion is octahedrally coordinated to the O1 and O9 oxygen atoms of the chromophore (CPH), and two water molecules act as the fifth and sixth ligands. The two coordinated water molecules are hydrogen bonded to O2 atoms of C5 and C13 bases. The Chro dimer binds at and significantly widens the minor groove of the GGCC sequence. The long axis of each chromophore lies along and stacks over the sugar-phosphate backbone with the two attached saccharide moieties (rings A/B and C/D/E) wrapping across the minor groove. DNA is kinked by 30 degrees and 36 degrees in the two complexes, respectively. Six G-specific hydrogen bonds between Chro and DNA provide the GGCC sequence specificity. Interestingly, DNA in concert with Chro appears to act as an effective template to catalyze the deamination of Co(NH3)6(3+), as shown by circular dichroism and crystal structure data. Our results present useful structural information for designing new anticancer drug derivatives in the future.

The potent anticancer drug actinomycin D (ActD) acts by binding to DNA, thereby interfering with replication and transcription. ActD inhibits RNA polymerase far more specifically than DNA polymerase. Such discrimination is not easily understood by the conventional DNA binding mode of ActD. We have solved and refined at 1.7 A resolution the crystal structure of ActD complexed to CGATCGATCG, which contains no canonical GpC binding sequence. The crystal data are space group P4(3)2(1)2, a = b = 47.01 A, and c = 160.37 A. The structure was solved by the multiple wavelength anomalous diffraction method using a 5-bromo-U DNA. The asymmetric unit of the unit cell contains two independent dimers of a novel slipped duplex complex consisting of two decamer DNA strands bound with two ActD drug molecules. (The DNA in one dimer is numbered C1 to G10 in one strand and C11 to G20 in the complementary strand and in the second dimer, C101 to G110 and C111 to G120, respectively.) The structure reveals a highly unusual ActD binding mode in which the DNA adopts a slipped duplex with the A3-T4/A13-T14 dinucleotides looped out. ActD intercalates between G2-C11* (C11* being from a symmetry-related molecule) and C5-G20 base pairs. Two such slipped duplex-ActD complexes bound to each other by mutually intercalating their T4/T14 bases into the helix cavities (located between C5-G20 and G6-C19 base pairs) of neighboring complexes, forming a dimer of drug-DNA complexes. The binding site mimics the drug binding at the elongation point during transcription. Modeling studies show that the ActD-DNA complex fits snugly in the active site cavity in RNA polymerase but not in DNA polymerase. This may explain the strong preference of ActD inhibition toward transcription.

Sso7d and Sac7d are two small chromatin proteins from the hyperthermophilic archaeabacterium Sulfolobus solfataricus and Sulfolobus acidocaldarius, respectively. The crystal structures of Sso7d-GTGATCGC, Sac7d-GTGATCGC and Sac7d-GTGATCAC have been determined and refined at 1.45 A, 2.2 A and 2.2 A, respectively, to investigate the DNA binding property of Sso7d/Sac7d in the presence of a T-G mismatch base-pair. Detailed structural analysis revealed that the intercalation site includes the T-G mismatch base-pair and Sso7d/Sac7d bind to that mismatch base-pair in a manner similar to regular DNA. In the Sso7d-GTGATCGC complex, a new inter-strand hydrogen bond between T2O4 and C14N4 is formed and well-order bridging water molecules are found. The results suggest that the less stable DNA stacking site involving a T-G mismatch may be a preferred site for protein side-chain intercalation., (Copyright 2000 Academic Press.)

The binding of a macrocyclic bisacridine and an antitumor intercalator ametantrone to DNA has been studied. We carried out X-ray diffraction analyses of the complexes between both intercalators and CGTACG. We have determined the crystal structure, by the multiple-wavelength anomalous diffraction (MAD) method, of bisacridine complexed with CGTA[br(5)C]G at 1.8 A resolution. The refined native crystal structure at 1.1 A resolution (space group C222, a = 29.58 A, b = 54.04 A, c = 40.22 A, and R-factor = 0.163) revealed that only one acridine of the bisacridine drug binds at the C5pG6 step of the DNA, with the other acridine plus both linkers completely disordered. Surprisingly, both terminal G.C base pairs are unraveled. The C1 nucleotide is disordered, and the G2 base is bridged to its own phosphate P2 through a hydrated Co(2+) ion. G12 is swung toward the minor groove with its base stacked over the backbone. The C7 nucleotide is flipped away from the duplex part and base paired to a 2-fold symmetry-related G6. The central four base pairs adopt the B-DNA conformation. An unusual intercalator platform is formed by bringing four complexes together (involving the 222 symmetry) such that the intercalator cavity is flanked by two sets of G x C base pairs (i.e., C5 x G8 and G6 x C7) on each side, joined together by G6 x G8 tertiary base pairing interactions. In the bisacridine-CGTACG complex, the intercalation platform is intercalated with two acridines, whereas in the ametantrone-CGTACG complex, only one ametantrone is bound. NMR titration of the bisacridine to AACGATCGTT suggests that the bisacridine prefers to bridge more than one DNA duplex by intercalating each acridine to different duplexes. The results may be relevant in understanding binding of certain intercalators to DNA structure associated with the quadruplet helix and Holliday junction.

G x A mismatched base pairs are frequently found in nucleic acids. Human centromere DNA sequences contain unusual repeating motifs, e.g. , (GAATG)n x (CATTC)n found in the human chromosome. The purine-rich strand of this repeating pentamer sequence forms duplex and hairpin structures with unusual stability. The high stability of these structures is contributed by the "sheared" G x A base pairs which present a novel recognition surface for ligands and proteins. We have solved the crystal structure, by the multiple-wavelength anomalous diffraction (MAD) method of d(CCGAATGAGG) in which the centromere core sequence motif GAATG is embedded. Three crystal forms were refined to near-atomic resolution. The structures reveal the detailed conformation of tandem G x A base pairs whose unique hydrogen-bonding surface has interesting interactions with bases, hydrated magnesium ions, cobalt(III)hexaammine, spermine, and water molecules. The results are relevant in understanding the structure associated with human centromere sequence in particular and G x A base pairs in nucleic acids (including RNA, like ribozyme) in general.

Structural studies are essential to understand mechanisms of non-sequence-specific DNA binding used by chromosomal proteins. A non-histone high-mobility group (HMG) chromosomal protein from Drosophila melanogaster, HMG-D, binds duplex DNA in a non-sequence-specific fashion. The DNA-binding domain of HMG-D has been co-crystallized with a duplex DNA fragment in the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell dimensions a = 43.74, b = 53.80, c = 86.84 A. Data have been collected to 2.20 A at 99 K, with diffraction observed to at least 2.0 A. Heavy-atom derivative crystals have been obtained by co-crystallization with oligonucleotides halogenated at major-groove positions near the end of the DNA.

A-DNA conformation is favored by guanine-rich sequences, such as (dG)n x (dC)n, or under low-humidity conditions. Earlier A-DNA crystal structures revealed some conformational variations which may be the result of sequence-dependent effects and/or crystal packing forces. Here we report the high-resolution crystal structure of d(AGGGGCCCCT) in two crystal forms (either in the P212121 or the P6122 space group) to gain insights into the conformation and dynamics of the (dG)n x (dC)n sequence. The P212121 form has been analyzed using data to 1.1 A resolution by the anisotropic temperature factor refinement procedure of the SHELX97 program. Such analysis affords us with the detailed geometric, conformational and motional property of an A-DNA structure. The backbone torsional angles fall in a narrow range, except for the alpha/gamma angles which have two distinct combinations (gauche-/gauche+ or trans/trans). An A-DNA model of poly(dG) x poly(dC) has been constructed using the conformational parameters derived from the crystal structure of the P212121 form. In the crystal structure of the P6122 space group, the central eight base pairs of the decamer adopt A-DNA conformation with the two terminal nucleotides flipped out to form base pairs with the neighboring nucleotides. Comparison of the A-DNA structure of the same sequence from two different crystal forms, reinforced the conclusion that molecules crystallized in the same space group have a more similar conformation, whereas the same molecule crystallized in different space groups has different (local) conformations.

This paper describes the progress in our efforts at producing ultra-high resolution (< 0.8 A) DNA structures using advanced cryo-crystallography and synchrotron. Our work is aimed at providing reliable geometric (bond length and bond angle), electronic and motional information of DNA molecules in different conformational contexts. These highly-reliable, new structures will be the basis for constructing better DNA force-field parameters, which will benefit the structural refinement of DNA, protein-DNA complexes, and ligand-DNA complexes.

The proteins Sac7d and Sso7d belong to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius and S. solfactaricus, respectively. These proteins are extremely stable to heat, acid and chemical agents. Sac7d binds to DNA without any particular sequence preference and thereby increases its melting temperature by approximately 40 degrees C. We have now solved and refined the crystal structure of Sac7d in complex with two DNA sequences to high resolution. The structures are examples of a nonspecific DNA-binding protein bound to DNA, and reveal that Sac7d binds in the minor groove, causing a sharp kinking of the DNA helix that is more marked than that induced by any sequence-specific DNA-binding proteins. The kink results from the intercalation of specific hydrophobic side chains of Sac7d into the DNA structure, but without causing any significant distortion of the protein structure relative to the uncomplexed protein in solution.

A-DNA is a stable alternative right-handed double helix that is favored by certain sequences (e.g., (dG)n.(dC)n) or under low humidity conditions. Earlier A-DNA structures of several DNA oligonucleotides and RNA.DNA chimeras have revealed some conformational variation that may be the result of sequence-dependent effects or crystal packing forces. In this study, four crystal structures of three decamer oligonucleotides, d(ACCGGCCGGT), d(ACCCGCGGGT), and r(GC)d(GTATACGC) in two crystal forms (either the P6(1)22 or the P2(1)2(1)2(1) space group) have been analyzed at high resolution to provide the molecular basis of the structural difference in an experimentally consistent manner. The study reveals that molecules crystallized in the same space group have a more similar A-DNA conformation, whereas the same molecule crystallized in different space groups has different (local) conformations. This suggests that even though the local structure is influenced by the crystal packing environments, the DNA molecule adjusts to adopt an overall conformation close to canonical A-DNA. For example, the six independent CpG steps in these four structures have different base-base stacking patterns, with their helical twist angles (omega) ranging from 28 degrees to 37 degrees. Our study further reveals the structural impact of different counter-ions on the A-DNA conformers. [Co(NH3)6]3+ has three unique A-DNA binding modes. One binds at the major groove side of a GpG step at the O6/N7 sites of guanine bases via hydrogen bonds. The other two modes involve the binding of ions to phosphates, either bridging across the narrow major groove or binding between two intra-strand adjacent phosphates. Those interactions may explain the recent spectroscopic and NMR observations that [Co(NH3)6]3+ is effective in inducing the B- to A-DNA transition for DNA with (G)n sequence. Interestingly, Ba2+ binds to the same O6/N7 sites on guanine by direct coordinations.

Among the new generations of anthracycline drugs, morpholino-doxorubicin (MDox) and its derivative have unusually potent activity when compared with the parent doxorubicin. 3"-Cyano-morpholino-doxorubicin (CN-MDox) has been suggested to form a covalent crosslink to DNA, although the exact mode of interactions remains unclear. To establish the structural basis of this crosslink, we carried out X-ray diffraction analyses of the complexes between four different morpholino-doxorubicins (i.e., MDox, CN-MDox, (R)- and (S)-2"-methoxy-morpholino-Dox (MMDox)) and two DNA hexamers CGTACG and CGATCG. Their crystal data are similar to other Dau/Dox complexes with space group P4(1)2(1)2,a = b approximately 28 A, c approximately 53 A. The refined structures at approximately 1.8 A resolution revealed that two drug molecules bind to the duplex with the aglycons intercalated between the CpG steps with their N3'-morpholino-daunosamines in the minor groove. The morpholino moiety is flexible and may adopt different conformations dependent on the sequence context. The O1" atoms of the two morpholino groups in the drug-DNA complexes are in van der Waals contact. The structural results suggest possible crosslinking mechanism of CN-MDox. It is worth pointing out that by linking two piperazinyl- or piperidinyl-doxorubicins at the 1" positions a new type of bis-doxorubicin derivatives may be synthesized which may bind to a hexanucleotide sequence with some specificity.

O6-ethyl-guanine (e6G) is a relatively persistent alkylation lesion caused by the exposure of DNA to carcinogen N-ethyl-N-nitrosourea. We have studied the structural consequences of the e6G incorporation in DNA by X-ray crystallography and NMR. We have obtained crystals of the modified DNA dodecamer d(CGC[e6G]AATTCGCG) complexed to several minor groove binding drugs including Hoechst 33258, Hoechst 33342, netropsin, and SN6999. The space group of the crystals from those complexes is P2(1)2(1)2(1). However the crystal structure of the SN6999 complex is not isomorphous to that from the other three complexes. In all four refined crystal structures the drugs bind in the narrow minor groove at or close to the central AATT region of the dodecamer B-DNA duplex. The DNA conformation is influenced by the binding of drugs. The eight independent e6G:C base pairs have a conformation ranging from one with three-centered hydrogen bonds between the bases to a wobble conformation with two hydrogen bonds. The ethyl group of the eight e6G bases is mostly in the proximal orientation to N7. Our 1D and 2D-NMR studies of the same (free) dodecamer reveal that the e6G:C base pairs in the duplex are likely to adopt a wobble conformation in solution. Those results suggest that the e6G:C base pair has a dynamic equilibrium among various conformations, which may present an ambiguous signal to cells. In contrast, the e6G:T base pair adopts a Watson-Crick-like conformation. This may be a plausible explanation of why thymine is found preferentially incorporated across the e6G during replication.

The interaction between a potent synthetic antitumor and antiviral minor groove binding drug 1-methyl-4-[4-[4-(4-(1-methylquinolinium)amino)benzamido]anilino] pyridinium dichloride (SN6999) and an alkylated DNA d(CGC[e6G]AATTCGCG) dodecamer has been studied by X-ray crystallography. The complex forms a new crystal lattice in the space group P2(1)2(1)2(1) with unit cell dimensions of a = 28.48 A, b = 36.11 A, and c = 69.60 A. The structure has been solved by the molecular replacement method and refined to an R-factor of 17.0% at approximately 2.5 A resolution using 1618 reflections. In the complex, the SN6999 covers almost six base pairs in the narrow minor groove with the 1-methylquinolinium (Q) ring near T8-A17 and the 1-methylpyridinium (P) ring near the C3-G22 base pair. The central benzamido (BQ) and anilino (BP) rings are essentially coplanar, with the Q and P rings having large dihedral angles of 38 degrees and 39 degrees, respectively, to the plane of BQ/BP. There is only one direct hydrogen bond between the amide NH of SN6999 to T20O2 of DNA. The drug-DNA interaction is stabilized by stacking interaction of sugar oxygens from T20O4' to BQ and C21O4' to BP. There is charge-induced dipole interaction between the positively charged nitrogen atom of 1-methylquinolinium with C9O4' and that of 1-methylpyridinium with G22O4'. The crystal structure of the complex can be used to explain the NMR results. SN6999 lacks the crescent shape observed in other minor groove binding drugs and distorts the DNA duplex upon binding. The complex packs in the lattice using the G-N2:G-N3 interlocking base pairs at both ends of the helix. As in earlier cases, the two independent e6G:C base pairs adopt different base pairing schemes. The e6G16:C9 base pair adopts a previously observed bifurcated configuration involving three-centered hydrogen bonds and is similar to a Watson-Crick pairing. In contrast, the e6G4:C21 base pair adopts a novel "reverse wobble" configuration with C21 being pushed toward the major groove side. The ethyl group is in the proximal orientation (to N7) in both base pairs. Taken together with the observations found in the same DNA complexed to Hoechst 33258, Hoechst 33342, and retropsin from different crystal lattices, the results suggest that the e6G:C base pairing is weak and polymorphic when compared to a normal G:C base pair and the DNA duplex containing this lesion is readily distorted.

Metal ion coordination to nucleic acids is not only required for charge neutralization, it is also essential for the biological function of nucleic acids. The structural impact of different metal ion coordinations of DNA helices is an open question. We carried out X-ray diffraction analyses of the interactions of the two transition metal ions Co(II) and Cu(II) and an alkaline earth metal ion Ba(II), with DNA of different conformations. In crystals, Co(II) ion binds exclusively at the N7 position of guanine bases by direct coordination. The coordination geometry around Co(II) is octahedral, although some sites have an incomplete hydration shell. The averaged Co-N7 bond distance is 2.3 A. The averaged Co-N7-C8 angle is 121 degrees, significantly smaller than the value of 128 degrees if the Co-N7 vector were to bisect the C5-N7-C8 bond angle. Model building of Co(II) binding to guanine N7 in B-DNA indicates that the coordinated waters in the axial positions would have a van der Waals clash with the neighboring base on the 5' side. In contrast, the major groove of A-DNA does not have enough room to accommodate the entire hydration shell. This suggests that Co(II) binding to either B-DNA or A-DNA may induce significant conformational changes. The Z-DNA structure of Cu(II)-soaked CGCGTG crystal revealed that the Cu(II) ion is bis-coordinated to N7 position of G10 and #G12 (# denotes a symmetry-related position) bases with a trigonal bipyramid geometry, suggesting a possible N7-Cu-N7 crosslinking mechanism. A similar bis-coordination to two guanines has also been seen in the interaction of Cu(II) in m5CGUAm5CG Z-DNA crystal and of Ba(II) with two other Z-DNA crystals.

Anthracycline antibiotics (notably daunorubicin (DAU) and doxorubicin (DOX)) and nucleoside analog arabinosylcytosine (araC or aC) are important anticancer drugs. They are sometimes used together in the treatment of certain cancers. Both classes of compounds act by blocking DNA replication and transcription. To probe whether both drugs can be incorporated simultaneously into DNA and the possible structural consequences, we carried out x-ray diffraction analyses of the complexes between DAU/DOX and araC-containing DNA hexamers cross-linked with formaldehyde. The crystal structures were determined to high resolution (DAU-CGaCGCG, 1.2 A, space group P4(1)2(1)2, R = 0.182, 3275 reflections; DOX-CAaCGTG, 1.5 A, space group C2, R = 0.175, 3359 reflections), and they are similar to those of the previously studied DAU- and DOX-DNA complexes, despite different crystal packings. Two DAU/DOX molecules intercalate at both ends of the helix with their amino sugars in the minor groove. As in the structure of DAU-CGCGCG (Wang, A.H.-J., Gao, Y.-G., Liaw, Y.-C., and Li, Y.K. (1991) Biochemistry 30, 3812-3815), a covalent methylene bridge (from formaldehyde) between the N3' of daunosamine and the N2 of the guanine is formed in both adducts. In DOX-CAaCGTG, the two halves are slightly different with a root-mean-square deviation of 0.322 A between them. The O14 hydroxyls of the intercalated DOXs are within hydrogen bond distances to the O2P atoms of the A2p(aC3) and A8p(AC9) steps. The O2'-hydroxyl group from araC does not affect the binding of DAU-DOX or the conformation of the drug-DNA complexes. The results suggest that three major drug modifications on DNA, i.e., intercalation, covalent bond formation, and nucleoside analog incorporation, can coexist in the same DNA molecule without difficulty. When they occur in close proximity in DNA, they may provide an additive inhibitory effect for the target enzymes.

Oligonucleotides with modification at the 5'-end have been used for various biochemical applications. As a first step to better assess the effects of those modifications on DNA conformation, we determined at 1.4-A resolution the left-handed Z-DNA structure of a DNA hexamer, aminohexyl-5'-d(pCpGp[br5C]pGpCpG), by X-ray diffraction analysis. This hexamer was crystallized in the monoclinic C2 (a = 51.13 A, b = 18.44 A, c = 34.67 A, and beta = 120.9 degrees) space group. Its structure has been refined by the restrained least-squares refinement to a final R factor of 0.164 using 3727 [> 2.0 sigma (F)] observed reflections. The overall conformation of the double helix resembles that of the canonical Z-DNA. The terminal 5'-phosphate groups of the dC residues adopt conformations (beta approximately 180 degrees and gamma approximately 60 degrees) similar to phosphodiester's conformation of the internal dC residues. Two types of interhelical stackings are observed, one of which may serve as a model for a single-strand nick in the backbone of DNA double helix. A barium ion is found to bridge two side-by-side Z-DNA helices by coordinating to the O6 and N7 atoms of two guanines simultaneously. This "cross-linking" ability of barium ion may be a useful property in promoting the reversible aggregation of nucleic acids.

Arabinosylcytosine (araC) is an important anticancer drug that has been shown to be misincorporated into DNA double helix. The incorporation of araC into DNA may have significant conformational consequences that could affect the function of DNA. In this paper, we present the high-resolution 3D structure of an araC-containing decamer d[CCAGGC(araC)TGG], as determined by X-ray diffraction analysis, and assess the possible DNA structural perturbation induced by araC. The modified decamer was crystallized in the monoclinic C2 (a = 31.97 A, b = 25.56 A, c = 34.62 A and beta = 114.50 degrees) space group, the same as that from d(CCAGGCCTGG) [Heinemann, U., & Alings, C. (1989) J. Mol. Biol. 210, 369]. The structure of the araC-containing decamer was solved by the molecular replacement method and refined by the constrained least-squares refinement procedure to obtain a final R factor of 0.187 using 2349 [greater than 2.0 sigma(F)] observed reflections to a resolution of 1.6 A. The overall conformation resembles that of the canonical decamer DNA structure, but with significant differences in regions close to the araC site. The O2' hydroxyl groups of the araC residues lie in the major groove of the helix, and they are in close contact with the C5 methyl and C6 H6 atoms of the thymine on the 3'-side. This creates a higher buckle in the araC7-G14 base pair (14 degrees), as compared to that found in the canonical decamer (9 degrees). This may slightly destabilize B-DNA. No direct intramolecular hydrogen bond is formed, in contrast to the situation when araC is incorporated into Z-DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular and crystal structure of steffimycin have been determined by single crystal X-ray diffraction to 0.9 angstrom resolution. The triclinic crystals are in the space group P1, with the unit cell dimensions of a = 8.606(3) angstrom, b = 22.168(7) angstrom, c = 8.448(2) angstrom, alpha = 97.56(3) degrees, beta = 95.97(2) degrees, gamma = 87.94(3) degrees, Z = 2. The structure was solved by direct methods and refined by the full-matrix least-squares method to a final R value of 0.065 with 3405 (Inet greater than 2.0 sigma (Inet] observed reflections using the NRCVAX software package. The crystal lattice includes 2 independent steffimycin, 3 water and one 2-methyl-2,4-pentanediol molecules. The conformation of steffimycin is grossly similar to other anthracycline antibiotics including daunorubicin. The crystal packing interactions of steffimycin suggest a preferred stacking of the aglycone chromophore of the antibiotic which resembles the intercalative interactions seen in the daunorubicin-d(CGTACG) (Wang et al., Biochemistry 26, 1152 (1987] and nogalamycin-d(CGT(pS)ACG) (Liaw et al., Biochemistry 28, 9913 (1989] complexes. The atomic coordinates data from these complexes were used to model the intercalative binding of steffimycin to DNA. The models were then stereochemically idealized by the constraint refinement program NUCLSQ. Subsequently XPLOR software package was used for energy minimization of these models in vacuo. The model building studies suggest that steffimycin has a higher CpG base sequence specificity over the TpA step, similar to that of daunorubicin and nogalamycin.

MAR70 is a synthetic derivative of the anticancer drug daunorubicin that contains an additional sugar, attached to the O4' of daunosamine. When MAR70 was crystallized with the DNA hexamer d(CGTnACG), where nA is 2-aminoadenine, a covalent methylene bridge was formed between the N3' of daunosamine and the N2 of 2-aminoadenine. This spontaneous reaction occurred through the crosslinking action of formaldehyde. The crosslink was demonstrated by the three-dimensional structure of the 2:1 adduct between MAR70 and d(CGTnACG) solved at 1.3-A resolution by x-ray diffraction analysis. The perfect juxtaposition of the two amino groups in the complex provides a template for efficient addition of formaldehyde. This adduct structure is compared with the analogous structure at 1.5-A resolution of the complex of MAR70-d(CGTACG), in which no formaldehyde addition was observed. In both complexes, two MAR70 molecules bind to the DNA hexamer double helix; the elongated aglycon chromophore is intercalated between the CpG steps and spans the G.C Watson-Crick base pairs. The disaccharides occupy nearly the entire minor groove of the distorted B-DNA hexamer double helix. The second sugar is in contact with the sugar-phosphate backbone and does not affect the binding interactions of the daunorubicin portion to DNA. The structure allows us to model the binding to DNA of drugs having more extensive oligosaccharides. In addition, it suggests that placing a reactive (e.g., alkylating) functional group at the N3' amino position of daunorubicin might be a fruitful route for designing anticancer drugs.

Formaldehyde (HCHO) cross-links the anticancer drug daunorubicin (DAU) to DNA efficiently. When DAU is mixed with DNA hexamers, d(CGCGCG) and d(CGTDCG), in the presence of HCHO, stable covalent adducts of DNA are formed, as shown by the HPLC analyses. The major adducts are identical with the materials in the respective crystals which can be readily obtained from the 1:1 mixture of DAU-d(CGCGCG) and DAU-d(CGTDCG) plus HCHO, but not from the solution without HCHO. The high-resolution (1.5 A) X-ray crystal structure of those adducts shows unambiguously that they contain a covalent methylene bridge between the N3' of daunosamine and the N2 of the guanine or 2-aminoadenine. The perfect juxtaposition of the two amino groups in the minor groove of the complex provides a template for an efficient addition of HCHO. The methylene bridge does not perturb the conformation of the drug-DNA complex, when compared to the structure of DAU-d(CGTACG). The results suggest new approaches for synthesizing a new type of potential anticancer drug by attaching a reactive (e.g., alkylating) functional group at the N3' amino position of daunorubicin/doxorubicin. The stable drug-DNA adduct may be useful as probes for other biological studies.

The three-dimensional molecular structures of the complexes between a novel antitumor drug nogalamycin and its derivative U-58872 with a modified DNA hexamer d[m5CGT(pS)Am5CG] have been determined at 1.7- and 1.8-A resolution, respectively, by X-ray diffraction analyses. Both structures (in space group P6(1)) have been refined with constrained refinement procedure to final R factors of 0.208 (3386 reflections) and 0.196 (2143 reflections). In both complexes, two nogalamycins bind to the DNA hexamer double helix in a 2:1 ratio with the elongated aglycon chromophore intercalated between the CpG steps at both ends of the helix. The aglycon chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. Most of the sugars remain in the C2'-endo pucker family, except three deoxycytidine residues (terminal C1, C7, and internal C5). All nucleotides are in the anti conformation. Specific hydrogen bonds are found in the complex between the drug and guanine-cytosine bases in both grooves of the helix. One hydroxyl group of the aminoglucose donates a hydrogen bond to the N7 of guanine, while the other receives a hydrogen bond from the N4 amino group of cytosine. The orientation of these two hydrogen bonds suggests that nogalamycin prefers a GC base pair with its aglycon chromophore intercalating at the 5'-side of a guanine (between NpG), or at the 3'-side of a cytosine (between CpN) with the sugars pointing toward the GC base pair. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through, suggesting that nogalamycin prefers GC sequences embedded in a stretch of AT sequences.

The three-dimensional molecular structures of the complexes between an interesting antitumor drug, nogalamycin, and two DNA hexamers, d[CGT(pS)ACG] and d[m5CGT(pS)Am5CG], were determined at high resolution by X-ray diffraction analyses. Two nogalamycins bind to the DNA double helix in a 2:1 ratio with the aglycon chromophore intercalated between the CpG steps at both ends of the helix. The nogalose and aminoglucose sugars lie in the minor and major grooves, respectively, of the distorted B-DNA double helix. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. Specific hydrogen bonds are found in the complex between the drug and guanine bases. We suggest that nogalamycin may prefer GC sequences embedded in a stretch of AT sequences.