(+)-CC-1065 as a probe for intrinsic and protein-induced bending of DNA

(+)-CC -1065 is biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. This antibiotic covalently modifies DNA by alkylation of N-3 of a adenine in the minor groove. As a Structural consequence of covalent modification of DNA, the helix axis id bent into the minor groove. The drug-induced bending of DNA has similarities to intrinsic. A-tract bending and the 3′ adenine of A-tracts shows a unique reactivity to alkylation by (+) -CC-1065. Upon covalent modification of A-tracts, the magnitude of bending is increased and helix is stiffened. Using high-field NMR, hydroxyl-radical footprinting and gel electrophoresis, the molecular basis for the high reactivity of the bonding sequence 5′ - AGTTA* (an asterisk indicates the covalent modification site) to (+)-CC-1065 has been shown to involve the inherent conformational flexibility of this sequence. Furthermore, these studies also demonstrate that after alkylation the drug-induced bending is focused over the TT region. By analogy with the junction bend model for A-tracts, a ‘truncated junction bend model’ is proposed for this structure. Last, the application of (+)-CC-1065 entrapped/induced bending of DNA as a probe for the Sp1-induced bending of the 21-base-pair repeat an Mu transpose bending of the att L3 sequence is described.     

Effect of the (+)-CC-1065-(N3-adenine)DNA adduct on in vitro DNA synthesis mediated by Escherichia coli DNA polymerase.

(+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-CC-1065 are due to the ability of this compound to covalently modify DNA. (+)-CC-1065 reacts with duplex DNA to form an N3-adenine DNA adduct which lies in the minor groove of the DNA helix overlapping with a 5-base-pair region. As a consequence of covalent modification with (+)-CC-1065, the DNA helix bends into the minor groove and also undergoes winding and stiffening [Lee, C.-S., Sun, D., Kizu, R., & Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 203-213]. In the studies described here, in which we have constructed site-directed DNA adducts on single-stranded DNA templates, we have shown that (+)-CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all show strong blocks against progression of Klenow fragment, E. coli DNA polymerase, and T4 DNA polymerase. The inhibition of bypass of drug lesions by polymerase could be partially alleviated by increasing the concentration of dNTPs and, to a small extent, by increasing polymerase levels. Klenow fragment binds equally well to a DNA template adjacent to a drug modification site and to unmodified DNA. These results taken together lead us to suspect that it is primarily inhibition of base pairing around the drug modification site and not prevention of polymerase binding that leads to blockage of DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the major tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA adduct by 1H and 15N NMR studies

(+)-CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of the drug are thought to be due to the formation of a covalent adduct with DNA through N3 of adenine. Although the covalent linkage sites between (+)-CC-1065 and DNA have been determined, the tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA duplex adduct was not defined. A [6-15N]deoxyadenosine-labeled 12 base pair non-self-complementary oligomer, d(GGCGGAGTT*AGG).d(CCTAACTCCGCC) (asterisk indicates 15N-labeled base), containing the (+)-CC-1065 most preferred binding sequence 5'AGTTA, was synthesized and modified with (+)-CC-1065. This [6-15N]deoxyadenosine-labeled 12-mer duplex adduct was then studied by 1H and 15N NMR. One-dimensional NOE difference and two-dimensional NOESY 1H NMR experiments on the nonisotopically labeled 12-mer duplex adduct demonstrate that the 6-amino protons of the covalently modified adenine exhibit two signals at 9.19 and 9.08 ppm. Proton NMR experiments on the [6-15N]deoxyadenosine-labeled 12-mer duplex adduct show that the two resonance signals for adenine H6 observed on the nonisotopically labeled duplex adduct were split into doublets by the 15N nucleus with coupling constants of 91.3 Hz for non-hydrogen-bonded and 86.8 Hz for hydrogen-bonded amino protons. Parallel 15N NMR experiments on the [6-15N]deoxyadenosine-labeled (+)-CC-1065-12-mer duplex adduct show a triplet-like signal around -276.9 ppm and coupling constants of 91.5 and 85.6 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the (+)-CC-1065-DNA adduct: critical role of ordered water molecules and implications for involvement of phosphate catalysis in the covalent reaction

(+)-CC-1065 is an extremely potent antitumor agent produced by Streptomyces zelensis. The potent effects of (+)-CC-1065 and its alkylating analogues are thought to be due to the formation of a covalent adduct through N3 of adenine in DNA. It has been previously postulated, on the basis of modeling studies, that a phosphate may be involved in stabilization of the adduct and in acid catalysis of this reaction. In this study, using 1H NMR in combination with 17O-labeled water and phosphate, we demonstrate the involvement of a bridging water molecule between a phenolic proton on the alkylating subunit of (+)-CC-1065 and an anionic oxygen in the phosphate on the noncovalently modified strand of DNA. In addition, a second ordered water molecule associated with one of the protons on N6 of the covalently modified adenine is also identified. This structure has important implications for catalytic activation of the covalent reaction between (+)-CC-1065 and DNA and, consequently, the molecular basis for sequence-selective recognition of DNA by the alkylating subunit of (+)-CC-1065. On the basis of the example described here, the use of 1H NMR in 17O-labeled water may be a powerful probe to examine other structures and catalytic processes for water-mediated hydrogen-bonded bridges that occur between small molecules and DNA or enzymes.     

Transposase contacts with mu DNA ends.

We have investigated the interaction between phage Mu transposase (A protein) and the ends (att sites) of Mu by chemical and nuclease protection and interference studies. These studies define a 24-base pair contact region at five of the six att sites (L1, L3 at att L and R1, R2, R3 at att R). Hydroxyl radical footprints show that the transposase binds to one face of the DNA helix and covers two consecutive major grooves. Binding specificity is achieved primarily through the major groove. Strong contacts are found with 3 guanines which are conserved at five of the sites. Two of these guanines are missing in the weakest binding site (L2) where 13 base pairs are mainly contacted. A pair of DNAase I hypersensitive sites, one on each strand, appear at the back of only one of the two contacted major grooves at most sites except at L2, and can be correlated with the degree of A protein-induced bend (Kuo, C.-F., Zou, A., Jayaram, M., Getzoff, E. D., and Harshey, R. M. (1991) EMBO J. 10, 1585-1591) at these sites. No contacts are observed for 4-5 base pairs in the vicinity of L1 and R1, where the A protein nicks DNA during transposition.     

Determination of the structural role of the linking moieties in the DNA binding of adozelesin

Adozelesin (formerly U73975, The Upjohn Co.) is a monofunctional DNA alkylating analogue of the antitumor antibiotic (+)-CC-1065. Adozelesin consists of a cyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one (CPI) alkylating subunit of (+)-CC-1065 and a indole and benzofurans subunit replacing the more complex pyrroloindole B and C subunits, respectively, of (+)-CC-1065. Previous studies have shown that adozelesin forms a reversible covalent DNA duplex adduct via a reaction between the N3 of adenine and the cyclopropyl of the cyclopropapyrroloindole (CPI) subunit. Gel electrophoresis studies have shown that adozelesin, like all the monofunctional (CPI)-based antitumor antibiotics, has a sequence preference for 5'-TTA-3' [the asterisk () indicates covalently modified base]. Molecular-modeling studies have shown that the bound adozelesin ligand spans a total of five base pairs including the modified adenine. These studies have also indicated that, owing to the orientation of the ligand within the base minor groove, there should be an overall preference for sequences rich in A.T base pairs, thus avoiding steric crowding around the exocyclic NH(2) of any guanines present. In this study, we have prepared and studied, by high-field NMR and restrained molecular mechanics (rMM) and dynamics (rMD), the duplex adduct formed between adozelesin and 5'-CGTAAGCGCTTACG-3'. Previous molecular-modeling studies suggested that this sequence should be less preferred, since the two GC base pairs should lead to extensive steric crowding within the adduct, and this hypothesis has, however, never been supported by DNA-footprinting data. (1)H NMR of the adozelesin duplex adduct has reveals that, although Watson-Crick base pairing is maintained throughout the DNA duplex, there is significant distortion around the central base pairs. This distortion is the result of strong hydrogen-bonding between the amide linker of the indole and benzofuran subunits, and the carbonyl of a central thymine base and second, weaker, hydrogen bond to the exocyclic NH(2) of the central guanine was also observed. (1)H NMR and rMD also indicate that, to accommodate this hydrogen-bond system, the bound adozelesin is not positioned centrally within the minor groove but pushed toward the modified DNA strand. Previous studies on the dimeric CPI analogue bizelesin have indicated the important role the ureylene linker plays in the DNA binding. This study indicates that a similar situation exists in the reaction of adozelesin with double-stranded DNA and provides a possible explanation into the unpredicted sequence selectivity of these ligands.     

Reaction of the antitumor antibiotic CC-1065 with DNA. Location of the site of thermally induced strand breakage and analysis of DNA sequence specificity

CC-1065 is a unique antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of this drug are thought to be due to its ability to form a covalent adduct with DNA through N3 of adenine. Thermal treatment of CC-1065-DNA adducts leads to DNA strand breakage. We have shown that the CC-1065 structural modification of DNA that leads to DNA strand breakage is related to the primary alkylation site on DNA. The thermally induced DNA strand breakage occurs between the deoxyribose at the adenine covalent binding site and the phosphate on the 3' side. No residual modification of DNA is detected on the opposite strand around the CC-1065 lesion. Using the early promoter element of SV40 DNA as a target, we have examined the DNA sequence specificity of CC-1065. A consensus sequence analysis of CC-1065 binding sites on DNA reveals two distinct classes of sequences for which CC-1065 is highly specific, i.e., 5'PuNTTA and 5'AAAAA. The orientation of the DNA sequence specificity relative to the covalent binding site provides a basis for predicting the polarity of drug binding in the minor groove. Stereo drawings of the CC-1065-DNA adduct are proposed that are predictive of features of the CC-1065-DNA adduct elucidated in this investigation.     

Mutational analysis of the att DNA-binding domain of phage Mu transposase.

The transposase (A protein) of phage Mu encodes binding to two families of DNA sites, att sites located at the Mu ends and enhancer sites located internally. Separate subdomains in the N-terminal domain I of Mu A protein are known to be involved in recognition of the att and enhancer sites. We have delineated an approximately 135 aa region within domain I beta gamma that specifies binding to Mu att sites. This peptide was overexpressed and its properties compared with that of the larger domain I beta gamma as well as the intact Mu A protein. Extensive mutagenesis of residues around a putative helix-turn-helix DNA-binding motif within the I beta domain identified several mutants defective in DNA transposition in vivo. Of these, Mu A(K157Q) was completely defective in att DNA-binding. Mu A(F131S) and Mu A(R146N) had a lower affinity for att DNA and low levels of transposition in vitro. Our results indicate that residues in the gamma region are required for activity and that residues outside the beta gamma region must also influence discrimination between the multiple att sites.     

Construction and characterization of a site-directed CC-1065-N3-adenine adduct within a 117 base pair DNA restriction fragment

The design, construction, and characterization of a site-directed CC-1065-N3-adenine adduct in a 117 base pair segment of M13mpI DNA are described. CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have demonstrated that the cyclopropyl ring of CC-1065 reacts quite specifically with N3 of adenine in double-stranded DNA to form a CC-1065-DNA adduct. Following alkylation, the drug molecule lies snugly within the minor groove of DNA, overlapping with five base pairs for which a marked sequence preference exists [Hurley, L. H., Reynolds, V. R., Swenson, D. H., Petzold, G. L., & Scahill, T. A. (1984) Science (Washington, D.C.) 226, 843-844]. On the basis of the unique characteristics of the reaction of CC-1065 with DNA and the structure of the resulting DNA adduct, we have designed a general strategy to construct a site-directed CC-1065-DNA adduct in a restriction fragment. The presence of unique AluI and HaeIII restriction enzymes sites on each side of a high-affinity CC-1065 binding sequence (5'-GATTA) permitted the preparation of a partial duplex DNA molecule containing the CC-1065 binding sequence in the duplex DNA region. Since CC-1065 only binds to duplex DNA, potential CC-1065 binding sequences in the long single-stranded regions were protected from drug binding during the construction process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the number and location of the manganese binding sites of DNA quadruplexes in solution by EPR and NMR.

The paramagnetic metal ion Mn2+ has been used to probe the electrostatic potentials of a DNA quadruplex that has two quartets with an overall fold of the chair type. A quadruplex with a basket type structure has also been examined. The binding of the paramagnetic ion manganese to these quadruplex DNAs has been investigated by solution state electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies. The EPR results indicate that the DNA aptamer, d(GGTTGGTGTGGTTGG), binds two manganese ions and that the binding constants for each of these sites is approximately 10(5) M-1. The NMR results indicate that the binding sites of the manganese are in the narrow grooves of this quadruplex DNA. The binding sites of the DNA quadruplex formed by dimers of d(GGGGTTTTGGGG) which forms a basket structure are also in the narrow groove. These results indicate that the close approach of phosphates in the narrow minor grooves of the quadruplex structures provide strong binding sites for the manganese ions and that EPR and NMR monitoring of manganese binding can be used to distinguish between the different types of quadruplex structures.     

Minor groove DNA alkylation directed by major groove triplex forming oligodeoxyribonucleotides.

We describe sequence-specific alkylation in the minor groove of double-stranded DNA by a hybridization-triggered reactive group conjugated to a triplex forming oligodeoxyribonucleotide (TFO) that binds in the major groove. The 24 nt TFOs (G/A motif) were designed to form triplexes with a homopurine tract within a 65 bp target duplex. They were conjugated to an N 5-methyl-cyclopropapyrroloindole (MCPI) residue, a structural analog of cyclopropapyrroloindole (CPI), the reactive subunit of the potent antibiotic CC-1065. These moieties react in the DNA minor groove, alkylating adenines at their N3 position. In order to optimize alkylation efficiency, linkers between the TFO and the MCPI were varied both in length and composition. Quantitative alkylation of target DNA was achieved when the dihydropyrroloindole (DPI) subunit of CC-1065 was incorporated between an octa(propylene phosphate) linker and MCPI. The required long linker traversed one strand of the target duplex from the major groove-bound TFO to deliver the reactive group to the minor groove. Alkylation was directed by relative positioning of the TFOs. Sites in the minor groove within 4-8 nt from the end of the TFO bearing the reactive group were selectively alkylated.     

Molecular basis for sequence-specific DNA alkylation by CC-1065

CC-1065 is a potent antitumor antibiotic that binds covalently to N3 of adenine in the minor groove of DNA. The CC-1065 molecule is made up of three repeating pyrroloindole subunits, one of which (the left-hand one or A subunit) contains a reactive cyclopropyl function. The drug reacts with adenines in DNA in a highly sequence-specific manner, overlapping four base pairs to the 5'-side of the covalently modified base. Concomitant with CC-1065 covalent binding to DNA is an asymmetric effect on local DNA structure which extends more than one helix turn to the 5'-side of the covalent binding site. The DNA alkylation, sequence specificity, and biological potency of CC-1065 and a select group of trimeric synthetic analogues were evaluated. The results suggest that (a) noncovalent interactions between this series of compounds and DNA do not lead to the formation of complexes stable enough to be detected by footprinting methods, (b) sequence specificity and alkylation intensity can be modulated by the substituents on the nonreactive middle and right-hand segments, and (c) biological potency correlates well with ability to alkylate DNA. In addition, the extent and the sequence specificity of covalent adduct formation between linear DNA fragments and three analogues comprised of the CC-1065 alkylating subunit linked to zero (analogue A), one (analogue AB), or two (analogue ABC) nonreactive indole subunits were compared.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-protein complexes during attachment-site synapsis in Mu DNA transposition.

Initial events in Mu DNA transposition involve specific recognition of Mu DNA ends (att sites) and an internal enhancer site by the Mu transposase (A protein). This interaction between A protein and Mu DNA sequences present on a supercoiled DNA substrate leads to the formation of a stable synaptic complex in which the att ends are nicked, prior to DNA strand transfer. This study examines the properties of a synaptic complex proficient for DNA transposition. We show that the A protein binds as a monomer to its binding sites, and causes the DNA to bend through approximately 90 degrees at each site. All six att binding sites (three at each Mu end) are occupied by A within the synaptic complex. Three of these sites are loosely held and can be emptied of A upon challenge with heparin. A synaptic complex with only three sites occupied is stable and is fully competent in the subsequent strand-transfer step of transposition.     

Linkers designed to intercalate the double helix greatly facilitate DNA alkylation by triplex-forming oligonucleotides carrying a cyclopropapyrroloindole reactive moiety.

Triplex-forming oligonucleotides (TFOs) bind sequence-specifically in the major groove of double-stranded DNA. Cyclopropapyrroloindole (CPI), the electrophilic moiety that comprises the reactive subunit of the antibiotic CC-1065, gives hybridization-triggered alkylation at the N-3 position of adenines when bound in the minor groove of double-stranded DNA. In order to attain TFO-directed targeting of CPI, we designed and tested linkers to 'thread' DNA from the major groove-bound TFO to the minor groove binding site of CPI. Placement of an aromatic ring in the linker significantly enhanced the site-directed reaction, possibly due to a 'threading' mechanism where the aromatic ring is intercalated. All of the linkers containing aromatic rings provided efficient alkylation of the duplex target. The linker containing an acridine ring system, the strongest intercalator in the series, gave a small but clearly detectable amount of non-TFO-specific alkylation. An equivalent-length linker without an aromatic ring was very inefficient in DNA target alkylation.     

Crystal structure of the excisionase-DNA complex from bacteriophage lambda

The excisionase (Xis) protein from bacteriophage lambda is the best characterized member of a large family of recombination directionality factors that control integrase-mediated DNA rearrangements. It triggers phage excision by cooperatively binding to sites X1 and X2 within the phage, bending DNA significantly and recruiting the phage-encoded integrase (Int) protein to site P2. We have determined the co-crystal structure of Xis with its X2 DNA-binding site at 1.7A resolution. Xis forms a unique winged-helix motif that interacts with the major and minor grooves of its binding site using an alpha-helix and an ordered beta-hairpin (wing), respectively. Recognition is achieved through an elaborate water-mediated hydrogen-bonding network at the major groove interface, while the preformed hairpin forms largely non-specific interactions with the minor groove. The structure of the complex provides insights into how Xis recruits Int cooperatively, and suggests a plausible mechanism by which it may distort longer DNA fragments significantly. It reveals a surface on the protein that is likely to mediate Xis-Xis interactions required for its cooperative binding to DNA.     

Altering the DNA-binding specificity of Mu transposase in vitro.

We describe the isolation of a variant of Mu transposase (MuA protein) which can recognize altered att sites at the ends of Mu DNA. No prior knowledge of the structure of the DNA binding domain or its mode of interaction with att DNA was necessary to obtain this variant. Protein secondary structure programs initially helped target mutations to predicted helical regions within a subdomain of MuA demonstrated to harbor att DNA binding activity. Of the 54 mutant positions examined, only two showed decreased affinity for att DNA, while eight others affected assembly of the Mu transpososome. A variant impaired in DNA binding [MuA(R146V)], and predicted to be in the recognition helix of an HTH motif, was challenged with altered att sites created from degenerate oligonucleotides to select for novel DNA binding specificity. DNA sequences bound to MuA(R146V) were detected by gel-retardation, and following several steps of PCR amplification/enrichment, were identified by cloning and sequencing. The strategy allowed recovery of an altered att site for which MuA(R146V) showed higher affinity than for the wild-type site, although this site was bound by wild-type MuA as well. The altered association between MuA(R146V) and an altered att site target was competent in transposition. We discuss the strengths and limitations of this methodology, which has applications in dissecting the functional role of specific protein-DNA associations.     

DNA-directed alkylating ligands as potential antitumor agents: sequence specificity of alkylation by intercalating aniline mustards

The sequence preferences for alkylation of a series of novel parasubstituted aniline mustards linked to the DNA-intercalating chromophore 9-aminoacridine by an alkyl chain of variable length were studied by using procedures analogous to Maxam-Gilbert reactions. The compounds alkylate DNA at both guanine and adenine sites. For mustards linked to the acridine by a short alkyl chain through a para O- or S-link group, 5'-GT sequences are the most preferred sites at which N7-guanine alkylation occurs. For analogues with longer chain lengths, the preference of 5'-GT sequences diminishes in favor of N7-adenine alkylation at the complementary 5'-AC sequence. Magnesium ions are shown to selectively inhibit alkylation at the N7 of adenine (in the major groove) by these compounds but not the alkylation at the N3 of adenine (in the minor groove) by the antitumor antibiotic CC-1065. Effects of chromophore variation were also studied by using aniline mustards linked to quinazoline and sterically hindered tert-butyl-9-aminoacridine chromophores. The results demonstrate that in this series of DNA-directed mustards the noncovalent interactions of the carrier chromophores with DNA significantly modify the sequence selectivity of alkylation by the mustard. Relationships between the DNA alkylation patterns of these compounds and their biological activities are discussed.     

1 A crystal structures of B-DNA reveal sequence-specific binding and groove-specific bending of DNA by magnesium and calcium

The 1 A resolution X-ray crystal structures of Mg(2+) and Ca(2+) salts of the B-DNA decamers CCAACGTTGG and CCAGCGCTGG reveal sequence-specific binding of Mg(2+) and Ca(2+) to the major and minor grooves of DNA, as well as non-specific binding to backbone phosphate oxygen atoms. Minor groove binding involves H-bond interactions between cross-strand DNA base atoms of adjacent base-pairs and the cations' water ligands. In the major groove the cations' water ligands can interact through H-bonds with O and N atoms from either one base or adjacent bases, and in addition the softer Ca(2+) can form polar covalent bonds bridging adjacent N7 and O6 atoms at GG bases. For reasons outlined earlier, localized monovalent cations are neither expected nor found.Ultra-high atomic resolution gives an unprecedented view of hydration in both grooves of DNA, permits an analysis of individual anisotropic displacement parameters, and reveals up to 22 divalent cations per DNA duplex. Each DNA helix is quite anisotropic, and alternate conformations, with motion in the direction of opening and closing the minor groove, are observed for the sugar-phosphate backbone. Taking into consideration the variability of experimental parameters and crystal packing environments among these four helices, and 24 other Mg(2+) and Ca(2+) bound B-DNA structures, we conclude that sequence-specific and strand-specific binding of Mg(2+) and Ca(2+) to the major groove causes DNA bending by base-roll compression towards the major groove, while sequence-specific binding of Mg(2+) and Ca(2+) in the minor groove has a negligible effect on helix curvature. The minor groove opens and closes to accommodate Mg(2+) and Ca(2+) without the necessity for significant bending of the overall helix.The program Shelxdna was written to facilitate refinement and analysis of X-ray crystal structures by Shelxl-97 and to plot and analyze one or more Curves and Freehelix output files.     

What drives proteins into the major or minor grooves of DNA?

The energetic profiles of a significant number of protein-DNA systems at 20 °C reveal that, despite comparable Gibbs free energies, association with the major groove is primarily an enthalpy-driven process, whereas binding to the minor groove is characterized by an unfavorable enthalpy that is compensated by favorable entropic contributions. These distinct energetic signatures for major versus minor groove binding are irrespective of the magnitude of DNA bending and/or the extent of binding-induced protein refolding. The primary determinants of their different energetic profiles appear to be the distinct hydration properties of the major and minor grooves; namely, that the water in the A+T-rich minor groove is in a highly ordered state and its removal results in a substantial positive contribution to the binding entropy. Since the entropic forces driving protein binding into the minor groove are a consequence of displacing water ordered by the regular arrangement of polar contacts, they cannot be regarded as hydrophobic.