DNA sequence recognition by the antitumor antibiotic CC-1065 and analogs of CC-1065

The DNA base pair preferences of the antitumor antibiotic CC-1065 and two analogs of CC-1065 were studied by following the rate of covalent bond formation (N-3 adenine adduct) with DNA oligomers containing the 5'NNTTA* and 5'NNAAA* sequences (N = nucleotide, A* = alkylated adenine). The rate of adduct formation of CC-1065 is greatly affected by DNA base changes at the fourth and fifth positions of the bonding site for the 5'NNAAA sequences, but not the 5'NNTTA sequences. However, an analog of CC-1065 containing the same alkylating moiety as CC-1065, but not the third fused ring system or additional methylene and oxygen substituents, shows similar rates of adduct formation for all sequences. A second analog of CC-1065 containing three fused ring systems, but not the methylene and oxygen substituents of CC-1065, shows rates of adduct formation with the same sequence dependence as CC-1065, but does not distinguish between the sequences to the degree shown by CC-1065. Adduct formation of CC-1065, but not the analogs, competes with a reversibly bound species. Thymine bases to the 3' side of a potentially reactive adenine or a cytosine base at the fifth position from the bonding adenine create reversible binding sites which decrease the rate of adduct formation of CC-1065. The sequence 5'GCGAATT binds CC-1065 only reversibly. This sequence can compete for CC-1065 with covalent bonding sequences if the sites are located in different oligomers, or if the sites are located (overlapped or not overlapped) in the same oligomer. The results of these competitive binding experiments suggest that the transfer of CC-1065 from the reversible binding site to the covalent bonding site with both sites located on a single DNA duplex, not overlapped, occurs through an equilibrium of CC-1065 in solution, not by migration of CC-1065 in the minor groove.     

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.     

Characterization of an adduct between CC-1065 and a defined oligodeoxynucleotide duplex.

CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. The drug binds covalently through N-3 of adenine and lies within the minor groove of DNA. Previous studies indicated that CC-1065 reacted with adenine in DNA to yield a thermally labile product that could be used to reveal its sequence specificity. These studies also provided insight into a DNA sequence (5'-CGGAGTTAGGGGCG-3') which should bind one molecule of CC-1065 in an unambiguous manner. This sequence, which contains the CC-1065 adenine binding site within the sequence 5'-TTA-3' was chemically synthesized together with the complementary strand. CC-1065 reacted with the oligoduplex to give an adduct that maintained the B-DNA form and had a final CD spectrum similar to those of the CC-1065 complexes formed with calf thymus DNA. The above 14mer was 5' end-labelled with 32P, annealed with its complementary strand, reacted with CC-1065 and heated. Drug-mediated strand breakage was evaluated on a sequencing gel. A single break occurred in the labelled strands to give a fragment that migrated as an 8.5mer; subsequent piperidine treatment produced a fragment that migrated as a 7mer, which is the size expected from the known binding of CC-1065 at adenine in 5'-TTA-3' sequences.     

Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065.

CC-1065 is a very potent antitumor antibiotic capable of covalent and noncovalent binding to the minor groove of naked DNA. Upon thermal treatment, covalent adducts formed between CC-1065 and DNA generate strand breaks [Reynolds, R. L., Molineux, I. J., Kaplan, D.J., Swenson, D.H., & Hurley, L.H. (1985) Biochemistry 24, 6228-6237]. We have shown that this molecular damage can be detected following CC-1065 treatment of mammalian whole cells. Using alkaline sucrose gradient analysis, we observe thermally induced breakage of [14C]thymidine-prelabeled DNA from drug-treated African green monkey kidney BSC-1 cells. Very little damage to cellular DNA by CC-1065 can be detected without first heating the drug-treated samples. CC-1065 can also generate heat-labile sites within DNA during cell lysis and heating, subsequent to the exposure of cells to drug, suggesting that a pool of free and noncovalently bound drug is available for posttreatment adduct formation. This effect was controlled for by mixing [3H]thymidine-labeled untreated cells with the [14C]thymidine-labeled drug-treated samples. The lowest drug dose at which heat-labile sites were detected was 3 nM CC-1065 (3 single-stranded breaks/10(6) base pairs). This concentration reduced survival of BSC-1 cells to 0.1% in cytotoxicity assays. The generation of CC-1065-induced lesions in cellular DNA is time dependent (the frequency of lesions caused by a 60 nM treatment reaching a plateau at 2 h) and is not readily reversible. The induction of heat-labile sites in cellular DNA was confirmed by gel electrophoretic analyses of the damage to intracellular simian virus 40 (SV40) DNA in SV40-infected BSC-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 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.     

Reversibility of the covalent reaction of CC-1065 and analogues with DNA.

Covalent DNA adducts of the antitumor antibiotic CC-1065 and its analogues undergo a retrohomologous Michael reaction in aqueous/organic solvent mixtures to regenerate the initial cyclopropylpyrroloindole (CPI) structure and, presumably, intact DNA. This reaction, which at higher temperatures competes with depurination of the N3-alkylated adenine, also occurs to a significant extent at 37 degrees C in neutral aqueous solution. Tritium-labeled adozelesin, covalently bonded to a 3-kilobase DNA restriction fragment which was exhaustively extracted to remove unbonded drug, was efficiently transferred to a 1-kilobase fragment upon coincubation for 20 h at 37 degrees C in aqueous buffer. Covalent adducts of adozelesin, but not CC-1065, on calf thymus DNA were cytotoxic to L1210 cells after incubation for 3 days at 37 degrees C, indicating that reversal of DNA alkylation can mediate potent cellular effects for simplified CC-1065 analogues.     

Recognition and repair of the CC-1065-(N3-adenine)-DNA adduct by the UVRABC nucleases

The recognition and repair of the helix-stabilizing and relatively nondistortive CC-1065-(N3-adenine)-DNA adduct by UVRABC nuclease has been investigated both in vivo with phi X174 RFI DNA by a transfection assay and in vitro by a site-directed adduct in a 117 base pair fragment from M13mp1. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis which binds within the minor groove of DNA through N3 of adenine. In contrast to the helix-destabilizing and distortive modifications of DNA caused by ultraviolet light or N-acetoxy-2-(acetylamino)fluorene, CC-1065 increases the melting point of DNA and decreases the S1 nuclease activity. Using a viral DNA-Escherichia coli transfection system, we have found that the uvrA, uvrB, and uvrC genes, which code for the major excision repair proteins for UV- and NAAAF-induced DNA damage, are also involved in the repair of CC-1065-DNA adducts. In contrast, the uvrD gene product, which has been found to be involved in the repair of UV damage, has no effect in repairing CC-1065-DNA adducts. Purified UVRA, UVRB, and UVRC proteins must work in concert to incise the drug-modified phi X174 RFI DNA. Using a site-directed and multiple CC-1065 modified (MspI-BstNI) 117 base pair fragment from M13mp1, we have found that UVRABC nuclease incises at the eighth phosphodiester bond on the 5' side of the CC-1065-DNA adduct on the drug-modified strand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depletion of nicotinamide adenine dinucleotide in normal and xeroderma pigmentosum fibroblast cells by the antitumor drug CC-1065

CC-1065 is an extremely potent antitumor antibiotic that forms a well-defined adduct with DNA in which the molecule lies within the minor groove and is covalently attached through N3 of adenine. Addition of CC-1065 to human fibroblast cells produced a prolonged depletion of the nicotinamide adenine dinucleotide (NAD) pool even at extremely low drug concentrations (0.01 microgram/mL). The depletion of NAD by CC-1065 was blocked by 3-aminobenzamide, which is consistent with a NAD depletion mechanism involving poly-(ADP-ribose) synthesis in response to a repair-induced DNA strand breakage event. Significantly, similar extents of NAD depletion were also evident in xeroderma pigmentosum cells of complementation groups A and D following exposure to CC-1065. Since this NAD depletion is presumably associated with repair-induced incision, the repair of CC-1065-DNA adducts can probably take place by a pathway distinct from that involved in repair of more conventional bulky DNA adducts. The prolonged depletion of NAD, even at low doses of drug, suggests that CC-1065 causes DNA damage that results in a delay or block in DNA excision repair between the excision and ligation steps.     

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)     

Single molecule linear analysis of DNA in nano-channel labeled with sequence specific fluorescent probes

An array of nano-channels was fabricated from silicon based semiconductor materials to stretch long, native dsDNA. Here we present a labeling scheme in which it is possible to identify the location of specific sequences along the stretched DNA molecules. The scheme proceeds by first using the strand displacement activity of the Vent (exo-) polymerase to generate single strand flaps on nicked dsDNA. These single strand flaps are hybridized with sequence specific fluorophore-labeled probes. Subsequent imaging of the DNA molecules inside a nano-channel array device allows for quantitative identification of the location of probes. The highly efficient DNA hybridization on the ss-DNA flaps is an excellent method to identify the sequence motifs of dsDNA as it gives us unique ability to control the length of the probe sequence and thus the frequency of hybridization sites on the DNA. We have also shown that this technique can be extended to a multi color labeling scheme by using different dye labeled probes or by combining with a DNA- polymerase-mediated incorporation of fluorophore-labeled nucleotides on nicking sites. Thus this labeling chemistry in conjunction with the nano-channel platform can be a powerful tool to solve complex structural variations in DNA which is of importance for both research and clinical diagnostics of genetic diseases.     

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.     

Theoretical study of the sequence specificity in the covalent binding of the antitumor drug CC-1065 to DNA

A theoretical modelling is presented of the covalent adducts of the antitumor agent CC-1065 with B-DNA. The optimal complexes are obtained by energy minimisation, taking into account full structure flexibility, including the flexible rings of the ligand and DNA. The binding preference of CC-1065 with respect to base sequence is studied. The results obtained elucidate the origin of the preference for two AT base pairs on the 5'side of the modified adenine. The modifications of the DNA structure upon ligand covalent binding are discussed.     

The antitumor agent CC-1065 inhibits helicase-catalyzed unwinding of duplex DNA.

The antitumor drug CC-1065 is thought to exert its effects by covalent bonding to N3 of adenine in DNA and interfering with some aspect of DNA metabolism. Therefore, it is of interest to determine what effect this drug has on enzymes involved in various aspects of DNA metabolism. In this report, we examine the ability of two DNA helicases, the dda protein of phage T4 and helicase II of Escherichia coli, to unwind CC-1065-adducted, tailed, oligonucleotides. It is shown that the presence of the drug on DNA strongly inhibits unwinding catalyzed by the T4 and E. coli proteins. A significant difference between the results obtained with the two helicases is that DNAs containing drug on either the tailed or the completely duplex strands are poor substrates for helicase II but dda protein-mediated unwinding is inhibited only when the drug is on the tailed strand. The drug-modified, helicase-released, strands migrate abnormally through a native gel, suggesting that the drug traps an unusual secondary structure generated in the course of protein-mediated unwinding. A kinetic analysis of the drug-inhibited reactions reveals that the helicases are trapped by the DNA-drug complex. This is evidenced by a decrease in the rate of helicase exchange between drug-bound substrate and drug-free duplex. The implications of these results with respect to the mechanism of action of CC-1065 in vivo are discussed.     

The binding of CC-1065 to thymidine and deoxyadenosine oligonucleotides and to poly(dA).poly(dT)

In this work, we report on the binding of the novel antitumor agent CC-1065 to poly(dA).poly(dT) and to mixtures of dA and dT oligomers as determined by electronic absorption and circular dichroism (CD) methods. In addition, the DNA binding properties of CC-1065 and its binding mechanism are compared to those of netropsin. CC-1065 binds to the polymer by at least three mechanisms to produce one irreversibly and two reversibly bound species. One reversibly bound species is moderately stable, but in time (days), it converts to the irreversibly bound species. Both of these species bind within the minor groove of the polymer and exhibit intense CC-1065 induced CD spectra. The other reversibly bound species does not acquire an induced CD. CC-1065 forces B-form duplex formation between mixtures of single strand dA and dT oligomers and binds irreversibly to the duplexes without showing the presence of an intermediate, reversibly bound species. The induced CD increases with increasing length of the oligomer, from the 5-mer (barely detectable CD) to the 14-mer (intense CD). The 7-, 10- and 14-mer mixtures bind about 1, between 1 and 2, and between 2 and 3 CC-1065 molecules, respectively. Computer graphic models of the CC-1065-DNA complex show that the covalent adduct of CC-1065 and unreacted CC-1065 can attain the same close van der Waals contacts between adenine C2 hydrogens and antibiotic CH groups that were observed in the crystal structure of the netropsin-DNA complex. These contacts may account for the dA-dT base pair binding specificity of CC-1065 and for the stability of the reversibly bound CC-1065 species.     

Calf thymus DNA binding/bonding properties of CC-1065 and analogs as related to their biological activities and toxicities.

CC-1065 is a potent natural antitumor antibiotic that binds non-covalently and covalently (N-3 adenine adduct) in the minor groove of B-form DNA. Synthetic analogs of CC-1065 do not exhibit the delayed death toxicity of CC-1065 and are efficacious anticancer agents, some of them curative in murine tumor models. In an attempt to understand the different biological properties of CC-1065 and analogs, we have determined the following quantities for CC-1065, enantiomeric CC-1065, and three biologically active analogs and their enantiomers: the calf thymus DNA (CT-DNA) induced molar ellipticity of the adduct (or how rigidly the adduct is held in the right-hand conformation of the minor groove); the stability of the adduct with respect to long incubation times and to digestion by snake venom phosphodiesterase I (SVPD); the stabilizing effect on the CT-DNA helix of the covalently and non-covalently bound species with respect to thermal melting; and the CT-DNA binding/bonding (non-covalent/covalent) profiles at a low molar ratio of nucleotide to drug. The major observations from these studies are as follows: (i) molecules which show large DNA interaction parameters, stable adducts, and significant non-covalent binding exhibit delayed death toxicity; (ii) molecules which show intermediate DNA interaction parameters and stable adducts, but do not show significant non-covalent binding, do not exhibit delayed death toxicity and are biologically active; (iii) molecules which show small DNA interaction parameters and unstable DNA adducts are biologically inactive. The results suggest that a window exists in the affinity for the minor groove of DNA wherein an analog may possess the correct balance of toxicity and activity to make a useful anticancer agent. Outside of this window, the analog causes delayed deaths or has no significant biological activity.     

Binding of CC-1065 to poly- and oligonucleotides

The binding of the antitumor agent CC-1065 to a variety of poly- and oligonucleotides was studied by electronic absorption, CD, and resistance to removal by Sephadex column chromatography. Competitive binding experiments between CC-1065 and netropsin were carried out with calf-thymus DNA, poly(dI-dC) · poly(dI-dC), poly(dI) · poly(dC), poly(rA) · poly(dT), poly(dA- dC) · poly(dG-dT), and poly(dA) · 2poly(dT). CC-1065 binds to polynucleotides by three mechanisms. In the first, CC-1065 binds only weakly, as judged by the induction of zero or very weak CD spectra and low resistance to extraction of drug from the polynucleotide by Sephadex chromatography. In the second and third mechanisms, CC-1065 binds strongly, as judged by the induction of two distinct, intense CD spectra and high resistance to extraction of drug from the polynucleotide, by Sephadex chromatography in both cases. The species bound by the second mechanism converts to that bound by the third mechanism with varying kinetics, which depend both on the base-pair sequence and composition of the polynucleotide. Competitive binding experiments with netropsin show that CC-1065 binds strongly in the minor groove of DNA by the second and third mechanisms of binding. Netropsin can displace CC-1065 that is bound by the second mechanism but not that bound by the third mechanism. CC-1065 binds preferentially to B-form duplex DNA and weakly (by the first binding mechanism) or not at all to RNA, DNA, and RNA–DNA polynucleotides which adopt the A-form conformation or to single-strand DNA. This correlation of strong binding of CC-1065 to B-form duplex DNA is consistent with x-ray data, which suggest an anomalous structure for poly(dI) · poly(rC), as compared with poly(rI) · poly(dC) (A-form) and poly(dI) · poly(dC) (B-form). The binding data indicate that poly(rA) · poly(dU) takes the B-form secondary structure like poly(rA) · poly(dT). Triple-stranded poly(dA) · 2poly(dT) and poly(dA) · 2poly(dU), which are considered to adopt the A-form conformation, bind CC-1065 strongly. Netropsin, which also shows a binding preference for B-form polynucleotides, also binds to poly(dA) · 2poly(dT) and occupies the same binding site as CC-1065. These binding studies are consistent with results of x-ray studies, which suggest that A-form triplex DNA retains some structural features of B-form DNA that are not present in A-form duplex DNA; i.e., the axial rise per nucleotide and the base tilt. Triple-stranded poly(dA) · 2poly(rU) does not bind CC-1065 strongly but has nearly the same conformation as poly(dA) · 2poly(dT) based on x-ray analysis. This suggests that the 2′-OH group of the poly(rU) strands interferes with CC-1065 binding to this polynucleotide. The same type of interference may occur for other RNA and DNA–RNA polynucleotides that bind CC-1065 weakly.     

ABC excinuclease incises both 5' and 3' to the CC-1065-DNA adduct and its incision activity is stimulated by DNA helicase II and DNA polymerase I

CC-1065 is a large molecule that binds covalently to adenine residues of DNA in a sequence-specific manner and lies in the minor groove about four bases to the 5' side of the adducted residue. Using a reconstituted Escherichia coli nucleotide excision repair system, we have obtained data showing that the ABC excinuclease makes incisions both 5' and 3' to the CC-1065 adduct and that the incision activity is stimulated by the addition of helicase II and DNA polymerase I (and dNTPs). Our results with the CC-1065 adduct are consistent with the reported in vitro processing of other adducts (e.g., cisplatin, UV photoproducts) but do not agree with a recent study that reported anomalous processing of the CC-1065 adduct by ABC excinuclease and helicase II. Our results also imply that, in binding to damaged DNA, ABC excinuclease does not make important contacts in the minor groove four bases to the 5' side of the damaged residue.     

An NMR study of the covalent and noncovalent interactions of CC-1065 and DNA

The binding of the antitumor drug CC-1065 has been studied with nuclear magnetic resonance (NMR) spectroscopy. This study involves two parts, the elucidation of the covalent binding site of the drug to DNA and a detailed investigation of the noncovalent interactions of CC-1065 with a DNA fragment through analysis of 2D NOE (NOESY) experiments. A CC-1065-DNA adduct was prepared, and an adenine adduct was released upon heating. NMR (1H and 13C) analysis of the adduct shows that the drug binds to N3 of adenine by reaction of its cyclopropyl group. The reaction pathway and product formed were determined by analysis of the 13C DEPT spectra. An octamer duplex, d(CGATTAGC.GCTAATCG), was synthesized and used in the interaction study of CC-1065 and the oligomer. The duplex and the drug-octamer complex were both analyzed by 2D spectroscopy (COSY, NOESY). The relative intensity of the NOEs observed between the drug (CC-1065) and the octamer duplex shows conclusively that the drug is located in the minor groove, covalently attached to N3 of adenine 6 and positioned from the 3'----5' end in relation to strand A [d(CGATTA6GC)]. A mechanism for drug binding and stabilization can be inferred from the NOE data and model-building studies.     

Sequence-dependent interactions of two forms of UvrC with DNA helix-stabilizing CC-1065-N3-adenine adducts

The uvrA, uvrB, and uvrC genes of Escherichia coli control the initial steps of nucleotide excision repair. The uvrC gene product is involved in at least one of the dual incisions produced by the UvrABC complex. Using single-stranded (ss) DNA affinity chromatography, we have separated two forms of UvrC from both wild-type E. coli cells and overproducing cells. UvrCI elutes at 0.4 M KCl, and UvrCII elutes at 0.6 M KCl. In general, both forms, in the presence of UvrA and UvrB, actively incise UV-irradiated and CC-1065-modified DNA in the same fashion; i.e., they incise six to eight nucleotides 5' to and three to five nucleotides 3' to a photoproduct or a CC-1065-N3-adenine adduct. They produce different incisions, however, at a CC-1065-N3-adenine adduct in the sequence 5'-GATTACG- present in the MspI-BstNI 117 bp fragment of M13mp1. UvrABCI incises at both the 5' and 3' sides of the adduct (UvrABCI cut), while UvrABCII incises only at the 5' side (UvrABCII cut). Mixing UvrCI and UvrCII results in both UvrABCI and UvrABCII cuts, and the levels of these two types of cutting are proportional to the amount of UvrCI and UvrCII. DNase I footprints of the MspI-BstNI 117 bp DNA fragment containing a site-directed CC-1065-adenine adduct at the 5'-GATTACG- site show that UvrCII, but not UvrCI, binds to the adduct site. Furthermore, the pattern of DNase I footprints induced by UvrCII binding differs from the pattern of the footprints induced by UvrA, UvrAB, and UvrABCI binding. Interestingly, while the presence of unirradiated DNA enhances the efficiency of UvrABCII in incising UV-irradiated DNA, it does not enhance UvrABCII incision of the CC-1065-N3-adenine adduct formed at 5'-GATTACG-. These results show that two different forms of UvrC differ in DNA binding properties as well as incision modes at some kinds of DNA damage.