7-Azido-Actinomycin D: A Photoaffinity Probe of the Sequence Specificity of DNA Binding by Actinomycin D

Abstract

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5′-dGpdC-3′ sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5′- flanking base was a pyrimidine and the 3′-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5′- TGGG-3′.     

1H and 31P NMR investigations of actinomycin D binding selectivity with oligodeoxyribonucleotides containing multiple adjacent d(GC) sites

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with adjacent 5'-GC-3' sites. ActD showed very high specificity for binding to GC sites regardless of oligomer length and surrounding sequence. For a first class of duplexes with a central GCGC sequence, a mixture of 1:1 complexes was observed due to the two different orientations of the ActD phenoxazone ring system. Analysis of 1H chemical shifts suggested that the favored 1:1 complex had the benzenoid side of the phenoxazone ring over the G base in the central base pair of the GCGC sequence. This is the first case in which an unsymmetrical intercalator has been shown to bind to DNA in both possible orientations. A unique 2:1 complex, with significantly different 1H and 31P chemical shifts relative to those of the 1:1 complexes, was formed with these same oligomers, again with the benzenoid side of the ActD molecule over the G base of the central GC base pair. There is considerable anticooperativity to binding of the second ActD in a GCGC sequence. In titrations of oligomers with the GCGC sequence, only the two 1:1 complexes are found up to ratios of one ActD per oligomer. Increasing the ActD concentration, however, resulted in stoichiometric formation of the unique 2:1 adduct. Spectrophotometric binding studies indicated that the apparent binding equilibrium constant for a GC site adjacent to a bound site is reduced by approximately a factor of 20 relative to the ActD binding constant to an isolated GC site.     

Photoaffinity approaches to determining the sequence selectivities of DNA-small molecule interactions: actinomycin D and ethidium.

The DNA photoaffinity ligands, 7-azidoactinomycin D and 8-azidoethidium, form DNA adducts that cause chain cleavage upon treatment with piperidine. Chemical DNA sequencing techniques were used to detect covalent binding. The relative preferences for modifications of all possible sites defined by a base pair step (e.g. GC) were determined within all quartet contexts such as (IGCJ). These preferences are described in terms of 'effective site occupations', which express the ability of a ligand to covalently modify some base in the binding site. Ideally, the effective site occupations measured for photoaffinity agents can also be related to site-specific, non-covalent association constants of the ligand. The sites most reactive with 7-azidoactinomycin D were those preferred for non-covalent binding of unsubstituted actinomycin D. GC sites were most reactive, but next-nearest neighbors exerted significant influences on reactivity. GC sites in 5'-(pyrimidine)GC(purine)-3' contexts, particularly TGCA, were most reactive, while reactivity was strongly suppressed for GC sites with a 5'-flanking G, or a 3'-flanking C. High reactivities were also observed for bases in the first (5') GG steps in TGGT, TGGG and TGGGT sequences recently shown to bind actinomycin D with high affinity. Pyrimidine-3',5'-purine steps and GG steps flanked by a T were most preferred by 8-azidoethidium, in agreement with the behavior of unsubstituted ethidium. The good correspondence between expected and observed covalent binding preferences of these two azide analogs demonstrates that photoaffinity labeling can identify highly preferred sites of non-covalent DNA binding by small molecules.     

Solution structure of the ActD-5'-CCGTT3GTGG-3' complex: drug interaction with tandem G.T mismatches and hairpin loop backbone

Binding of actinomycin D (ActD) to the seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3 GTGG-3' has been studied in solution using high-resolution nuclear magnetic resonance (NMR) techniques. A strong binding constant (8 x 10(6) M(-1)) and high quality NMR spectra have allowed us to determine the initial DNA structure using distance geometry as well as the final ActD-5'-CCGTT3 GTGG-3' complex structure using constrained molecular dynamics calculations. The DNA oligomer 5'-CCGTT3GTGG-3' in the complex forms a hairpin structure with tandem G.T mismatches at the stem region next to a loop of three stacked thymine bases pointing toward the major groove. Bipartite T2O-GH1 and T2O-G2NH2 hydrogen bonds were detected for the G.T mismatches that further stabilize this unusual DNA hairpin. The phenoxazone chromophore of ActD intercalates nicely between the tandem G.T mismatches in essentially one major orientation. Additional hydrophobic interactions between the ActD quinoid amino acid residues with the loop T5-T6-T7 backbone protons were also observed. The hydrophobic G-phenoxazone-G interaction in the ActD-5'-CCGTT3GTGG-3' complex is more robust than that of the classical ActD- 5'-CCGCT3GCGG-3' complex, consistent with the roughly 2-fold stronger binding of ActD to the 5'-CCGTT3GTGG-3' sequence than to its 5'-CCG CT3GCGG-3' counterpart. Stabilization by ActD of a hairpin containing non-canonical stem base pairs further strengthens the notion that ActD or other related compounds may serve as a sequence- specific ssDNA-binding agent that inhibits human immunodeficiency virus (HIV) and other retroviruses replicating through ssDNA intermediates.     

An NMR investigation of the binding of the anticancer drug actinomycin D to oligodeoxyribonucleotides with isolated 5'd(GC)3' binding sites

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with one GC binding site [d(ATATGCATAT) (1), d-(ATACGCGTAT) (2), and d(ATATACGCGTATAT) (3)] and with two GC sites [d(ATGCATGCAT) (4)]. At R = 1 (molar ratio of ActD to oligomer duplex) ActD caused a doubling of the number of imino proton signals at, and adjacent to, the GC binding site of 1. One of the G.C base pair signals shifted upfield while the other shifted downfield. Both of the signals for the A.T base pairs adjacent to the binding site shifted downfield. All imino proton signals of 2 and the longer sequence, 3, shifted upfield on binding of ActD to the GC site, indicating a sequence-dependent change in base stacking on complex formation. For both 1 and 2 addition of ActD resulted in a similar pattern of three downfield 31P NMR signals. The two most downfield signals have chemical shift and temperature dependence which are characteristic of phosphate groups at isolated intercalation sites. At R = 1 the ActD complex with 4 has very complex spectra with both upfield and downfield A.T and G.C imino signals. All these data were consistent with two 1:1 complexes with the unsymmetrical phenoxazone ring adopting both of the two possible orientations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of the aflatoxin B1-DNA adduct at N7 of guanine. Theoretical intercalation and covalent adduct models

Two theoretical models are proposed for the conformational structure of both intercalated and covalent adduct complexes of aflatoxin B1, designated AFB1, with N7 of guanine of DNA. The covalent adduct model requires the DNA to kink a minimum of 39 degrees about the covalent site of the C8 and N7 atoms comprising the bond of the covalent complex. The preference of AFB1 for specific G bases within a sequence of GC content followed that of experimental studies with the added feature that for binding to the third G base of a tetramer sequence from the 3'-end, the AFB1 displayed enhanced binding at the 3' site of the targeted guanosine. Binding of AFB1 to the second G base of a tetramer sequence from the 3'-end leads to preference for a 5' site of the targeted guanosine. Inhibition of AFB1's interaction with the targeted DNA in the presence of intercalated ethidium bromide is explained by these proposed models.     

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.     

2D NMR investigation of the binding of the anticancer drug actinomycin D to duplexed dATGCGCAT: conformational features of the unique 2:1 adduct

One- and two-dimensional NMR studies on the oligomer dA1T2G3C4G5C6A7T8, with and without actinomycin D (ActD), were conducted. Analysis of the NMR data, particularly 2D NOE intensities, revealed that the free oligonucleotide is a duplex in a standard right-handed B form. At the ratio of 1 ActD/duplex (R = 1), 1D NMR studies indicate that two 1:1 unsymmetric complexes form in unequal proportions with the phenoxazone ring intercalated at a GpC site, in agreement with previous studies [Scott, E.V., Jones, R.L., Banville, D.L., Zon, G., Marzilli, L.G., & Wilson, W.D. (1988) Biochemistry 27, 915-923]. The 2D COSY data also confirm this interpretation since eight cytosine H6 to H5 and two ActD H8 to H7 cross-peaks are observed. At R = 2, both COSY and NOESY spectra confirm the formation of a unique 2:1 species with C2 symmetry. The oligomer remains in a right-handed duplex but undergoes extreme conformational changes both at and adjacent to the binding site. The deoxyribose conformation of T2, C4, and C6 shifts from primarily C2'-endo in the free duplex to an increased amount of C3'-endo in the 2:1 complex as revealed by the greater intensity of the base H6 to 3' NOE cross-peak relative to the intensity of the H6 to H2' NOE cross-peak. This conformational change widens the minor groove and should help alleviate the steric crowding of the ActD peptides. The orientation of the ActD molecules at R = 2 has the quinoid portion of the phenoxazone ring at the G3pC4 site and the benzenoid portion of the phenoxazone ring at the G5pC6 site on the basis of NOE cross-peaks from ActD H7 and H8 to G5H8 and C6H6. All base pairs retain Watson-Crick type H-bonding, unlike echinomycin complexes [e.g., Gao, X., & Patel, D.J. (1988) Biochemistry 27, 1744-1751] where Hoogsteen base pairs have been observed. In contrast to previous studies on ActD, we were able to distinguish the two peptide chains.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of the principal promoter sequence of the c-H-ras transforming oncogene: deletion analysis of the 5''-flanking region by focus formation assay.

A number of deletion mutants were isolated, including 5', 3', and internal deletions in the 5'-flanking region of the human cellular oncogene related to the Harvey sarcoma virus (c-H-ras), and their transforming activities were examined in NIH 3T3 cells. DNA sequences which could not be detected without losing transforming activity were localized to a relatively short stretch upstream of the region which showed homology to the 5'-flanking region of v-H-ras oncogene. S1 nuclease analysis indicated that there were two clusters of mRNA start sites at positions that were about 1,371 and 1,298 base pairs upstream of the first coding ATG. The minimum region required for promoter function was estimated to be a 51-base-pair-long (or less) DNA segment. The promoter was GC rich (78%) and did not contain the consensus sequences that are usually observed in PolII-directed promoters but contained a GC box within which one of the mRNA start sites was included. In addition, two sets of positive and negative elements seemed to be located between the promoter and the protein-coding region, which appeared to influence positively and negatively, respectively, the efficiency of transformation with the c-H-ras oncogene.     

Molecular recognition between oligopeptides and nucleic acids. Specificity of binding of a monocationic bis-furan lexitropsin to DNA deduced from footprinting and 1H NMR studies

MPE-Fe(EDTA) footprinting of a novel monocationic bis-furan lexitropsin 6 on a HindIII/EcoRI restriction fragment of pBR322 DNA revealed a series of four-base binding sites (all 5'----3') of (primary) TGTA, TGAA, AAAT, ACAA, TTAT, and (secondary) CTAA, TCGT, TGTA, GTCA, and GGTT. Thus 6 can accept a GC pair at positions 1, 2 or 3 of the binding site with a strict 3' (4 position) AT requirement. Marked enhancement of cleavage, particularly at GC rich sequences, is observed at regions flanking or even up to 18 base pairs remote from a given binding site. The non-exchangeable and imino 1H NMR resonances of the 1:1 complex and d-[CATGGCCATG]2 were assigned using a combination of NOE differences, NOESY and COSY techniques. 1H NMR studies (ligand induced chemical shifts and NOE differences) of Lexitropsin 6 with d-[CATGGCCATG]2 show unambiguously the location and orientation of the N to C termini of 6 on the sequence 5'-G5C6C7A8-3', with the C terminus oriented to A8. This orientation of 6 in the minor groove of 5'-GCCA is confirmed by an NOE observed between H1 2a of 6 and AH8(8). This preference for binding of 6 to the sequence 5'-GCCA when challenged with d-[CATGGCCATG]2 is in accord with the conclusions of the footprinting experiments wherein GC base pairs can be accepted in the first three positions and with a strict 3' terminus AT reading requirement. Collectively the data support the inference of a GC recognizing capacity for a 2,5-substituted furan moiety within a lexitropsin. The 1H NMR data indicate that the decadeoxyribonucleotide duplex exists in the B conformation in both the 1:1 complex and the free form. The apparent binding constant of 6 to calf thymus DNA is 1.68 X 10(5) M-1 whereas netropsin under similar conditions gives a value of 1.85 X 10(7) M-1. This suggests that if advantage is to be taken of the GC recognizing property of a 2,5-substituted furan in longer lexitropsins it should be flanked by more strongly bound moieties.     

Interaction of a tissue-specific factor with an essential rat growth hormone gene promoter element.

Rat growth hormone (rGH) gene expression is normally restricted to the anterior pituitary. As a model of this tissue specificity, we compared the transient expression of an rGH-chloramphenicol acetyltransferase (CAT) hybrid gene in rGH-producing rat pituitary tumor (GC) cells and in non-rGH-producing rat fibroblast (rat-2) cells. Deletion analysis of the rGH portion of this hybrid gene demonstrated that DNA sequences within 140 base pairs 5' to the rGH gene were sufficient for correct cell type-specific expression. Deletion of an additional 35 base pairs of the rGH 5'-flanking DNA resulted in a loss of expression of the transfected hybrid gene and correlated with the interaction of a putative trans-acting factor with this region of the rGH promoter. This factor was detectable by DNase I footprinting in a crude nuclear extract from GC cells but not from rat-2 cells. Site-directed mutagenesis of the footprint region caused complete loss of expression of a hybrid gene containing 530 base pairs 5' to the rGH gene. Thus, the interaction of this factor, which we term GC2, is likely to be essential for the tissue-specific expression of the rGH gene.