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It is shown that the averaged chemical shift (ACS) of a particular nucleus in the protein backbone empirically correlates well to its secondary structure content (SSC). Chemical shift values of more than 200 proteins obtained from the Biological Magnetic Resonance Bank are used to calculate ACS values, and the SSC is estimated from the corresponding three-dimensional coordinates obtained from the Protein Data Bank. ACS values of (1)H(alpha) show the highest correlation to helical and sheet structure content (correlation coefficient of 0.80 and 0.75, respectively); (1)H(N) exhibits less reliability (0.65 for both sheet and helix), whereas such correlations are poor for the heteronuclei. SSC estimated using this correlation shows a good agreement with the conventional chemical shift index-based approach for a set of proteins that only have chemical shift information but no NMR or x-ray determined three-dimensional structure. These results suggest that even chemical shifts averaged over the entire protein retain significant information about the secondary structure. Thus, the correlation between ACS and SSC can be used to estimate secondary structure content and to monitor large-scale secondary structural changes in protein, as in folding studies.

The C-terminal regions of several DNA repair and cell cycle checkpoint proteins are homologous to the breast-cancer-associated BRCA-1 protein C-terminal region. These regions, known as BRCT domains, have been found to mediate important protein-protein interactions. We produced the BRCT domain of DNA ligase IIIalpha (L3[86]) for biophysical and structural characterization. A glutathione S-transferase (GST) fusion with the L3[86] domain (residues 837-922 of ligase IIIalpha) was expressed in Escherichia coli and purified by glutathione affinity chromatography. The GST fusion protein was removed by thrombin digestion and further purification steps. Using this method, (15)N-labeled and (13)C/(15)N-double-labeled L3[86] proteins were prepared to enable a full determination of structure and dynamics using heteronuclear NMR spectroscopy. To obtain evidence of binding activity to the distal BRCT of the repair protein XRCC1 (X1BRCTb), as well as to provide insight into the interaction between these two BRCT binding partners, the corresponding BRCT heterocomplexes were also prepared and studied. Changes in the secondary structures (amount of helix and sheet components) of the two constituents were not observed upon complex formation. However, the melting temperature of the complex was significantly higher relative to the values obtained for the L3[86] or X1BRCTb proteins alone. This increased thermostability imparted by the interaction between the two BRCT domains may explain why cells require XRCC1 to maintain ligase IIIalpha activity.

This paper reports on the combined NMR-molecular mechanics computational studies of the solution structure of the (-)-cis-anti-[BP]dG adduct positioned opposite dC in the sequence context d(C1- C2-A3-T4-C5-[BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14- A15-G16-C17-G18-A19-T20- G21-G22) duplex [designated (-)-cis-anti-[BP]dG.dC 11-mer duplex]. This adduct is derived from cis addition at C10 of (-)-anti-7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] to the N2 position of dG6 in this duplex sequence. The exchangeable and nonexchangeable protons of the benzo[a]pyrenyl moiety and nucleic acid of the major conformation were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. There was a general broadening of proton resonances for a three-nucleotide segment centered about the lesion site which resulted in a tentative assignment for the sugar protons of the C7 residue in the spectrum of the adduct duplex. The solution conformation of the major conformation of the (-)-cis-anti-[BP]dG.dC 11-mer duplex has been determined by incorporating DNA-DNA and intermolecular BP-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results establish that the covalently attached benzo[a]pyrenyl ring intercalates between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs. The modified deoxyguanosine [BP]-dG6 and its partner cytosine dC17 are looped out of the helix into the major groove. The purine ring of the [BP]dG6 residue is directed toward the 5'-end of the modified strand and stacks over the major groove edge of its 5'-side neighbor dC5 residue. The solution structure of the (-)-cis-anti-[BP]dG.dC 11-mer duplex is compared with those of the stereoisomeric (+)-trans-anti-[BP]dG [Cosman, M., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918], (-)-trans-anti-[BP]dG [de los Santos, C., et al. (1992) Biochemistry 31, 5245-5252], and (+)-cis-anti-[BP]dG [Cosman, M., et al. (1993a) Biochemistry 32, 4146-4155] adducts positioned opposite dC in the same duplex sequence context. A key finding is that the long axes of the intercalated benzo[a]pyrenyl rings in the solution structures of the (+)- and (-)- cis-anti-[BP]dG.dC 11-mer duplexes are oriented in opposite directions with the benzylic ring directed toward the minor groove in the (+)-cis isomer and toward the major groove in the (-)-cis isomer. In addition, a comparison is also made with the solution structure of the (+)-trans-anti-[BP]dG adduct opposite a deletion site [Cosman, M., et al. (1994a) Biochemistry 33, 11507-11517] since this adduct duplex displays several conformational features in common with the structure of the (-)-cis-anti-[BP]dG.dC 11-mer duplex. The structures of both duplex adducts exhibit intercalation of the covalently attached ligand into the helix and displacement of the modified deoxyguanosine into the major groove. Studies of the biological activities of stereochemically defined BP-DNA adducts and the comparison of the solution structure of the (-)-cis-anti-[BP]dG.dC 11-mer duplex with its stereoisomeric counterparts should lead to new insights into the relationships between defined helical distortions and mutagenic specificity and activity.

This paper reports on the solution structure of the [AF]dG adduct positioned opposite a deletion site in a DNA oligomer duplex that defines the alignment of the covalent aminofluorene--C8-guanine adduct relative to the deletion site. The combined NMR molecular mechanics computational studies were undertaken on the [AF]dG adduct embedded in the d(C5-[AF]G6-C7).d(G16-G17) sequence context in a duplex containing 11 residues on the modified strand and 10 on the partner, with no base opposite the modification. The exchangeable and nonexchangeable protons of the aminofluorene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution conformation of the [AF]G.del 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The aminofluorene ring of [AF]dG6 is intercalated between intact Watson-Crick dC5.dG17 and dC7.dG16 base pairs with the guanine base of [AF]dG6 in a syn alignment displaced into the major groove. The syn glycosidic torsion angle at [AF]dG6 is supported by both carbon and proton chemical shift data for the sugar resonances of the modified guanine residue. The long axis of the aminofluorene ring is parallel to the long axis of the flanking dG.dC base pairs with the AF ring undergoing rapid 180 degrees flips on the NMR time scale. The intercalation site is wedge shaped with a pronounced propeller-twisting and buckling of the dC5.dG17 base pair. The guanine base of [AF]dG6, which is positioned in the major groove, is inclined relative to the helix axis and stacks over the 5'-flanking dC5 residue in the solution structure. The intercalative-base displacement structure of the [AF]dG.del 11-mer duplex exhibits several unusually shifted proton resonances that can be readily accounted for by the ring current contributions of the guanine purine and carcinogen fluorene aromatic rings of the [AF]dG6 adduct. There are similarities between this structure of the AF-C8-dG covalent adduct positioned opposite a deletion site and the (+)-trans-anti-BP-N2-dG covalent adduct positioned opposite a deletion site in the same sequence context reported previously from this laboratory [Cosman et al. (1994) Biochemistry 33, 11507-11517]. The chromophores are intercalated into the helix opposite the deletion site with displacement of the modified guanine into the major groove in both cases.(ABSTRACT TRUNCATED AT 400 WORDS)

Therapies using antibodies directed against cell surface proteins have improved survival for human patients with non-Hodgkin's lymphoma (NHL). It is possible that similar immuno-therapeutic approaches may also benefit canine NHL patients. Unfortunately, variability between human and canine epitopes often limits the usefulness of such therapies in pet dogs. The Lym-1 antibody recognizes a unique epitope on HLA-DR10 that is expressed on the majority of human B-cell malignancies. The Lym-1 antibody has now been observed to bind to dog lymphocytes and B-cell NHL. Sequence comparisons and computer modeling of a human and three canine DRB1 proteins identified several orthologs of human HLA-DR10 expressed by dog lymphocytes. Immuno-staining confirmed the presence of proteins containing the Lym-1 epitope on dog lymphocytes and B-cell NHL. In addition, a selective high affinity ligand (SHAL) SH-7139 designed to bind within the Lym-1 epitope of HLA-DR10 was also observed to bind to canine B-cell NHL tissue. This SHAL, which is selectively cytotoxic to cells expressing HLA-DR10 and has been shown to cure mice bearing human B-cell lymphoma xenografts, may prove useful in treating B-cell malignancies in pet dogs.

Here we present modeling and NMR spectroscopic evidence that the function of a Yersinia pestis pMT1 plasmid protein, designated as orf38, is most likely a glutamine binding protein. The modeling was homology-based at a very low level of sequence identity ( approximately 16%) and involved structural comparison of multiple templates, as well as template-substrate interaction analyses. Transferred nuclear Overhauser and saturation transfer difference experiments were used to characterize the binding of sugars and amino acids to orf38. The identification and characterization of an unknown protein function using the strategy presented here has applicability to a variety of research areas, including functional genomics and proteomics efforts.

Selective high-affinity ligands (SHALs) were selected as substitutes for monoclonal antibodies (mAbs) to deliver radioisotopes to malignant tumors. Because a SHAL (5 KD) is considerably smaller in comparison to an antibody (150 KD), a significant therapeutic index (TI) enhancement for radioimmunotherapy (RIT) is anticipated. The antibody-antigen (Ab-Ag) model system chosen for the development of SHALs consists of Lym-1, a MAb with proven selectivity in non-Hodgkin's lymphoma (NHL) patients and its well-characterized Ag, the beta subunit of HLA DR10. Whereas Lym-1 is readily available, the subunit of HLA-DR10 is not. Native, heterodimeric (alpha and beta subunits) HLA-DR10 can be purified from Raji cells, which are known to overexpress this Ag. Inconsistent homogeneity between preparations of HLA-DR10 solubilized in the presence of detergents prompted us to express a recombinant form of the beta subunit of HLA-DR10 in Escherichia coli. Negligible production yields (or=50 microg/L) were achieved by the expression of the full-length protein in a soluble form. By contrast, yields of 240 mg/L were obtained by expressing only the extracellular domain (ED) of the beta subunit of HLA-DR10 in an insoluble form (inclusion bodies). The recovery yield of refolded protein was 75%. Circular dichroism (CD) and Lym-1 binding studies indicated that the recombinant ED of the beta subunit of HLA-DR10 was properly folded. Therefore, this recombinant protein can be used as a surrogate for native heterodimeric HLA DR10 for the in vitro selection of SHALs and related targeting molecules.

Aberrant association of autoantibodies with myelin oligodendrocyte glycoprotein (MOG), an integral membrane protein of the central nervous system (CNS) myelin, has been implicated in the pathogenesis of multiple sclerosis (MS). Sensitization of nonhuman primates (Callithrix jacchus marmosets) against the nonglycosylated, recombinant N-terminal domain of rat MOG (residues 1-125) reproduces an MS-like disease in which MOG-specific autoantibodies directly mediate demyelination. To assess the interrelationship between MOG structure and the induction of autoimmune CNS diseases and to enable structure-based rational design of therapeutics, a homology model of human MOG(2-120) was constructed based on consensus residues found in immunoglobulin superfamily variable-type proteins having known structures. Possible sites for posttranslational modifications and dimerization have also been identified and analyzed. The B cell and T cell epitopes have been identified in rat MOG-immunized marmosets, and these sequences are observed to map primarily onto accessible regions in the model, which may explain their ability to generate potent antibody responses.

BRCT (BRCA1 carboxyl terminus) domains are found in a number of DNA repair enzymes and cell cycle regulators and are believed to mediate important protein-protein interactions. The DNA ligase IIIalpha BRCT domain partners with the distal BRCT domain of the DNA repair protein XRCC1 (X1BRCTb) in the DNA base excision repair (BER) pathway. To elucidate the mechanisms by which these two domains can interact, we have determined the solution structure of human ligase IIIalpha BRCT (L3[86], residues 837-922). The structure of L3[86] consists of a beta2beta1beta3beta4 parallel sheet with a two-alpha-helix bundle packed against one face of the sheet. This fold is conserved in several proteins having a wide range of activities, including X1BRCTb [Zhang, X. D., et al. (1998) EMBO J. 17, 6404-6411]. L3[86] exists as a dimer in solution, but an insufficient number of NOE restraints precluded the determination of the homodimer structure. However, 13C isotope-filtered and hydrogen-deuterium exchange experiments indicate that the N-terminus, alpha1, the alpha1-beta2 loop, and the three residues following alpha2 are involved in forming the dimer interface, as similarly observed in the structure of X1BRCTb. NOE and dynamic data indicate that several residues (837-844) in the N-terminal region appear to interconvert between helix and random coil conformations. Further studies of other BRCT domains and of their complexes are needed to address how these proteins interact with one another, and to shed light on how mutations can lead to disruption of function and ultimately disease.

Site-specifically modified oligonucleotides were obtained in milligram quantities by reacting racemic 3t,4r-dihydroxy-1,2t-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (B[c]PhDE-2, or anti-B[c]PhDE) with the single deoxyadenosine (dA) residue in the oligodeoxynucleotide d(CTCTCACTTCC). Enzyme digestion of the covalently modified oligonucleotides with the exonuclease spleen phosphodiesterase yielded covalently linked B[ca]PhDE-N6-deoxyadenosyl monophosphate (dAMP) adducts. Comparisons of the reverse phase HPLC retention times and CD spectra of these B[c]PhDE-3'-dAMP mononucleotide adducts, with those of standards derived from the reaction of the enantiomers (+)- and (-)-anti-B[c]PhDE with 3'-dAMP, show that two major oligonucleotide adducts (I and II) were obtained upon reacting racemic anti-B[c]PhDE with d(CTCTCACTTCC). In oligonucleotide adduct I, the lesion is a (+)-trans-anti-B[c]PhDE-N6-dA residue, and in oligonucleotide adduct II it is a (-)-trans-anti-B[c]PhDE-N6-dA residue. These assignments were further confirmed using a standard 32P postlabeling assay of B[c]PhDE-3'-dAMP mononucleotide adducts obtained from the digestion of oligonucleotides I and II by spleen phosphodiesterase. The melting points (Tm) of duplexes of modified oligonucleotides I and II and their natural complementary strands are not affected significantly by the presence of the covalently bound benzo[c]phenanthrenyl residues. Opposite stereoselective resistance to enzyme digestion by the exonucleases snake venom phosphodiesterase and spleen phosphodiesterase is exhibited by the stereoisomeric (+)-trans- and (-)-trans-anti-B[c]PhDE-modified oligonucleotide adducts I and II; these results are consistent with the intercalative insertion of the benzo[c]phenanthrenyl residues on the 5'-side of the modified dA residue in adduct I, and its insertion on the 3'-side of the dA residue in adduct II, as observed in the duplexes by high resolution NMR techniques [Cosman et al. (1993) Biochemistry 32, 12488-12497, and Cosman et all, Biochemistry, in press.

This paper reports on NMR-molecular mechanics structural studies of the (-)- trans-anti-benzo[c]phenanthrene-dA adduct positioned opposite dT in the sequence context of the d(C1-T2-C3-T4-C5-[BPh]A6-C7-T8-T9-C10-C11).d(G12- G13-A14-A15-G16-T17-G18-A19-G20-A21- G22) duplex (designated as the (-)-trans-anti-[BPh]dA.dT 11-mer duplex). This adduct is derived from the covalent binding of (-)-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene [(-)-anti-BPhDE] to N6 of dA6 in this duplex sequence. The benzo[c]phenanthrenyl and nucleic acid exchangeable and nonexchangeable protons were assigned in the predominant conformation following analysis of two-dimensional NMR data sets in H2O and D2O buffer solution. The solution structure of the (-)-trans-anti-[BPh]dA.dT 11-mer duplex has been determined by incorporating intramolecular and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results show that the [BPh]dA6.dT17 base pair propeller twists and buckles slightly to permit the covalently attached benzo[c]phenanthrenyl ring to intercalate between the [BPh]dA6.dT17 and dC7.dG16 base pairs to the 3'-side of the [BPh]dA6 lesion site without disrupting the Watson-Crick hydrogen bond alignments in the modified duplex. The strain in the highly sterically hindered fjord region of the benzo[c]phenanthrenyl moiety is relieved by the propeller-like nonplanar geometry of the aromatic phenanthrenyl ring system, which stacks predominantly with the dG16 and dT17 bases on the unmodified strand. The benzylic ring adopts a distorted half-chair form, in which the H1 and H2 protons are pseudo-diequatorial and the H3 and H4 protons are pseudodiaxial. The current observation that the (-)-trans-anti-[BPh]dA positioned opposite dT intercalates to the 3'-side of the intact modified base pair contrasts with our previous demonstration that the stereoisomeric (+)-trans-anti-[BPh]dA adduct positioned opposite dT intercalates to the 5'-side of the intact modified base pair [Cosman, M., et al. (1993b) Biochemistry 32, 12488-12497]. These stereochemically induced structural differences between isomeric [BPh]dA lesions derived from the binding of chiral (+)- and (-)-anti-BPhDE enantiomers may in turn profoundly influence the interactions of the carcinogen-modified DNA with repair and replication enzymes in the cell.

This paper reports on the solution structure of the (+)-trans-anti-[BP]dG adduct positioned opposite a deletion site in a DNA oligomer duplex which defines the alignment of this covalent benzo[a]pyrene-N2-deoxyguanosine stereosiomer relative to the deletion site. The combined NMR-molecular mechanics computation studies were undertaken on the (+)-trans-anti-[BP]dG adduct embedded in the d(C5-[BP]G6-C7).d(G16-G17) sequence context in a duplex containing 11 residues on the modified strand and 10 on the partner, with no base opposite the modification. The exchangeable and nonexchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution conformation of the (+)-trans-anti-[BP]dG.del 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The benzo[a]pyrene ring of [BP]dG6 is intercalated between intact Watson-Crick dC5.dG17 and dC7.dG16 base pairs with the deoxyguanosine base of [BP]dG6 displaced into the major groove. The intercalation site is wedge shaped, being narrower toward the dG16-dG17 step on the deletion-containing strand. The deoxyguanosine base of [BP]dG6 which is positioned in the major groove is inclined relative to the helix axis and stacks over the 5'-flanking dC5 residue in the solution structure. The intercalative-base displacement structure of the (+)-trans-anti-[BP]dG.del 11-mer duplex exhibits several unusually shifted proton resonances which can be readily accounted for by the ring current contribution of the deoxyguanosyl and pyrenyl rings of the [BP]dG6 adduct. This solution structure of the (+)-trans-anti-[BP]dG.del 11-mer duplex where the pyrene ring intercalates into the helix with displacement of the modified deoxyguanosine into the major groove strikingly contrasts with our previous study on the (+)-trans-anti-[BP]dG.dC 11-mer duplex [Cosman, M., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918] where the benzo[a]pyrene ring is positioned in the minor groove without disruption of the Watson-Crick pairing at the [BP]dG.dC modification site. Thus, generation of the deletion site following removal of the dC opposite the (+)-trans-anti-[BP]dG results in a displacement of the entire [BP]dG residue toward the major groove and intercalation of the benzo[a]pyrene ring into the helix.

We have applied a combined NMR-molecular mechanics approach to determine the solution structure of the (+)-cis-anti-[BP]dG adduct positioned opposite a deletion site in the sequence d(C5-[BP]G6-C7).d(G16-G17) at the DNA oligomer duplex level. Our structural studies establish that the benzo[a]pyrene ring intercalates into the helix opposite the deletion site while the modified deoxyguanosine is displaced into the minor groove with its plane parallel to the helix axis. The intercalation site is wedge-shaped with the benzo[a]pyrene ring stacked over intact flanking Watson-Crick dG.dC base pairs. The modified deoxyguanosine stacks over the minor groove face of the sugar ring of the 5'-flanking dC5 residue. The structure at the lesion site is consistent with the observed intermolecular NOEs which served as input restraints to guide the molecular mechanics calculations, and, in addition, the various stacking interactions explain the observed large ring current shifts associated with adduct formation. The solution structure of the (+)-cis-anti-[BP]dG adduct positioned opposite a deletion site reported in this study is similar to the corresponding structure of the same adduct positioned opposite dC reported previously [Cosman, M., de los Santos, C., Fiala, R., Hingerty, B. E., Luna, E., Harvey, R. G., Geacintov, N. E., Broyde, S.,Patel, D. J. (1993) Biochemistry 32, 4145-4155]. This is not surprising since the dC opposite the lesion site was looped out of the helix and it can be readily replaced by a deletion site through minor changes associated with buckling of the intercalation site. By contrast, the solution structures of the (+)-trans-anti-[BP]dG and (+)-cis-anti-[BP]dG adducts positioned opposite deletion sites in the same sequence context exhibit distinct surface topologies in the grooves of the DNA helix. Thus, even though the benzo[a]pyrene intercalates into the helix opposite the deletion site in both cases, the modified deoxyguanosine is displaced into the major groove for the (+)-trans-anti-[BP]dG adduct while it is displaced into the minor groove for the (+)-cis-anti-[BP]dG stereoisomer. The orientational differences reflect the chiral characteristics of the two [BP]-dG stereoisomeric adducts with the different alignments of the bulky DNA lesion opposite the deletion site likely to influence interactions with the cellular repair machinery.

Benzo[c]phenanthrene diol epoxide can covalently bind to the exocyclic amino group of deoxyadenosine to generate [BPh]dA adducts where the polycyclic aromatic hydrocarbon is attached to the major groove edge of DNA. This paper reports on NMR-energy minimization structural studies of the (+)-trans-anti-[BPh]dA adduct positioned opposite dT in the sequence context d(C5-[BPh]A6-C7).d-(G16-T17-G18) at the 11-mer duplex level. The exchangeable and nonexchangeable protons of the benzo[c]phenanthrenyl moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution structure of the (+)-trans-anti-[BPh]dA.dT 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY data sets as restraints in energy minimization computations. The covalently attached benzo[c]phenanthrene ring intercalates to the 5'-side of the [BPh]-dA6 lesion site without disruption of the flanking Watson-Crick dC5.dG18 and [BPh]dA6.dT17 base pairs. The observed buckling of the intercalation cavity reflects the selective overlap of the intercalated phenanthrenyl ring with dT17 and dG18 bases on the unmodified strand. The structure provides new insights into how a polycyclic aromatic hydrocarbon covalently attached to the major groove edge of deoxyadenosine can still unidirectionally intercalate into the helix without disruption of the modified base pair. Our study establishes that among the contributing factors are a propeller-twisted [BPh]dA6.dT17 base pair, displacement of the carcinogen-DNA linkage bond from the plane of the dA6 base, the specific pucker adopted by the benzylic ring, and the propeller-like nonplanar geometry for the aromatic phenanthrenyl ring system. Our combined experimental-computational studies to date have now identified three structural motifs adopted by covalent polycyclic aromatic hydrocarbon-DNA adducts with their distribution determined by the chiral characteristics of individual stereoisomers and by whether the covalent adducts are generated at the minor or the major groove edge of the helix.

The deoxyribooligonucleotide 5'-d(CTCACATGTACACTCT) was reacted separately with the chiral diol epoxide isomers 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE)] and 7 alpha, 8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [(-)-anti-BPDE)], to produce the modified oligonucleotides 5'-d(CTCACATGBPDETACACTCT). Adducts in which either (+)-anti-BPDE or (-)-anti-BPDE are covalently bound via their C10 positions by trans addition to the exocyclic amino group of the single G residues were isolated and purified by HPLC methods. Snake venom phosphodiesterase (SVPD, phosphodiesterase I), which hydrolyzes DNA from the 3'-OH terminus to the 5'-end, digests the (+)-trans-anti-BPDE-oligonucleotide adducts at a significantly faster rate than that of the sterically different (-)-trans-anti-BPDE-oligonucleotide adducts. However, using spleen phosphodiesterase (SPD, phosphodiesterase II), which hydrolyzes DNA in the 5'-->3' direction, the opposite stereoselective resistance to digestion is observed. Using shorter BPDE-modified oligonucleotides as standards, the enzyme stall sites have been defined by gel electrophoresis methods; the most digestion-resistant phosphodiester linkage is the 5'-d(...T-G*...)-3' bond in the case of (+)-trans-BPDE-modified oligonucleotide adducts for both enzymes, SVPD and SPD (the starred G denotes the site of BPDE modification). In the case of the (-)-trans-BPDE-modified oligonucleotide adducts, the phosphodiester bond on the 3'-side of the modified G [5'-d(...G*-T...)-3'] is most resistant to digestion by both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

This paper reports on the solution structure of the (+)-cis-anti-[BP]dG adduct positioned opposite dC in a DNA oligomer duplex which provides the first experimentally based solution structure of an intercalative complex of a polycyclic aromatic hydrocarbon covalently bound to the N2 of deoxyguanosine. The combined NMR-energy minimization computation studies were undertaken on the (+)-cis-anti-[BP]dG adduct embedded in the same d(C5-[BP]G6-C7).d(G16-C17-G18) trinucleotide segment of the complementary 11-mer duplex studied previously with the stereoisomeric trans adducts. The exchangeable and nonexchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution structure of the (+)-cis-anti-[BP]dG-dC 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY data sets as restraints in energy minimization computations. The benzo[a]pyrene ring of [BP]dG6 is intercalated between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs in a right-handed DNA helix. The benzylic ring is in the minor groove while the pyrenyl ring sacks with flanking dC5 and dC7 bases on the same strand. The deoxyguanosine ring of [BP]dG6 is not Watson-Crick base paired but displaced into the minor groove with its plane parallel to the helix axis and stacks over the sugar ring of dC5. The dC17 base on the partner strand is displaced from the center of the helix toward the major groove by the intercalated benzo[a]pyrene ring. This intercalative structure of the (+)-cis-anti-[BP]dG-dC 11-mer duplex exhibits several unusually shifted proton resonances which can be readily accounted for by the ring current contributions of the deoxyguanosine and pyrenyl rings of the [BP]dG6 adduct. Several phosphorus resonances are shifted to low and high field of the unperturbed phosphorus spectral region and have been assigned to internucleotide phosphates centered about the [BP]dG6 modification site. These studies define the changes in the helix at the central trinucleotide segment needed to generate the intercalation site for the covalently bound (+)-cis-anti-[BP]dG adduct.(ABSTRACT TRUNCATED AT 400 WORDS)

We have synthesized, separated, and purified approximately 10 mg of a deoxyundecanucleotide duplex containing a single centrally positioned covalent adduct between (+)-anti-benzo[a]pyrene (BP) diol epoxide and the exocyclic amino group of guanosine. Excellent proton NMR spectra are observed for the (+)-trans-anti-BP diol epoxide-N2-dG adduct positioned opposite dC and flanked by G.C pairs in the d[C1-C2-A3-T4-C5-(BP)G6-C7-T8-A9-C10-C11].d[12- G13-T14-A15-G16-C17-G18-A19-T20-G 21-G22] duplex +ADdesignated (BP)G.C 11-mer+BD. We have determined the solution structure centered about the BP covalent adduct site in the (BP)G.C 11-mer duplex by incorporating intramolecular and intermolecular proton-proton distance bounds deduced from the NMR data sets as constraints in energy minimization computations. The BP ring is positioned in the minor groove and directed toward the 5' end of the modified strand. One face of the BP ring of (BP)G6 is stacked over the G18 and A19 sugar-phosphate backbone on the partner strand and the other face is exposed to solvent. A minimally perturbed B-DNA helix is observed for the d[T4-C5-(BP)G6-C7-T8].d[A15-G16-C17-G18-A19] segment centered about the adduct site with Watson-Crick alignment for both the (BP)G6.C17 pair and flanking G.C pairs. A widening of the minor groove at the adduct site is detected that accommodates the BP ring whose long axis makes an angle of approximately 45 degrees with the average direction of the DNA helix axis. Our study holds future promise for the characterization of other steroisomerically pure adducts of BP diol epoxides with DNA to elucidate the molecular basis of structure-activity relationships associated with the stereoisomer-dependent spectrum of mutational and carcinogenic activities.

The highly tumorigenic isomer (+)-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] and its non-tumorigenic enantiomer (-)-anti-BPDE are known to react predominantly with the exocyclic amino group (N2) of deoxyguanine in DNA and to form adducts of different conformations. The spectroscopic characteristics (UV absorbance, fluorescence and circular dichroism) of stereochemically defined (+)-trans, (-)-trans, (+)-cis and (-)-cis d(5'-CACATGBPDETACAC) adducts in the single-stranded form, or complexed with the complementary strand d(5'-GTGTACATGTG) in aqueous solution, were investigated. The spectroscopic characteristics of the double-stranded d(5'-CACATGBPDETACAC).d(5'-GTGTACATGTG) adducts can be interpreted in terms of two types of conformations. In site I-type conformations, there is an approximately 10 nm red shift in the absorption maxima, which is attributed to significant pyrenyl residue-base interactions; in site II-type adducts, the red shift is only approximately 2-3 nm, and the pyrene ring system is located at external, solvent-exposed binding sites. The spectroscopic characteristics of the BPDE-modified duplexes are of the site II type for the (+)- and (-)-trans, and of the site I type for the (+)- and (-)-cis adducts. In adducts derived from the binding of (+)-anti-BPDE to poly(dG-dC).(dG-dC) and poly(dG).(dC), the trans/cis BPDE-N2-dG adduct ratio is 6 +/- 1; in the case of (-)-anti-BPDE this ratio is only 0.4 +/- 0.1 and 0.6 +/- 0.15 in poly(dG-dC).(dG-dC) and poly(dG).(dC) respectively. The spectroscopic properties of these BPDE-modified polynucleotide adducts are consistent with those of the BPDE-modified oligonucleotide complexes; the cis adducts are correlated with site I adduct conformations, while the trans adducts are of the site II type. The correlations between adduct characteristics and biological activities of the two BPDE enantiomers are discussed.

A recently developed methodology [Jankowiak, R., Lu, P., Small, G. J., and Geacintov, N. E. (1990) Chem. Res. Toxicol. 3, 39-46], which combines fluorescence line narrowing spectroscopy at 4.2 K with non-line-narrowed (S2----S0 laser excitation) fluorescence spectroscopy at 77 K and fluorescence quenching, is used to characterize adducts formed from (+)-anti-BPDE and the alternating copolymers poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), the nonalternating poly (dG).poly(dC), single-strand poly(dG), and the oligonucleotide d(ATATGTATA). Detailed comparisons of the fluorescence spectra and quenching (with acrylamide) of the properties of the adducts with those of (+)-anti-BPDE-DNA adducts are made. Fluorescence spectra of the trans and cis isomers of the adduct formed from guanosine monophosphate and the adducts of d(ATATGTATA) are used to assign the stereochemistry of the two major DNA adducts as trans-N2-dG moieties which occupy two different DNA sites. Evidence for the existence of minor cis-type guanine adducts is provided. Finally, a fourth type of DNA adduct (minor) is identified and assigned as trans-N6-dA.

The reaction of the (+)- and (-)-enantiomers of BDPE trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) with the oligodeoxynucleotide d(ATATGTATA) in aqueous buffer solutions gives rise predominantly to trans and cis addition products at the exocyclic amino group of the single deoxyguanosine residue. The trans/cis ratios are 7:1 in the case of (+)-BPDE, and 2:1 in the case of (-)-BPDE, while the reaction yields correspond to 34 and 15% respectively, of modified strands. These relatively high reaction efficiencies, at least for this particular type of oligonucleotide sequence, offer the possibilities of synthesizing relatively large amounts of well-defined covalent BPDE-oligonucleotide adducts (with different sequences of nucleotides flanking the modified base) for detailed spectroscopic and biochemical studies.