Arthur Grollman | SUNY: Stony Brook University (original) (raw)
Papers by Arthur Grollman
Journal of Medicinal Chemistry, 1986
A series of simple aza and diaza bicyclic quinones related to the AB ring system of streptonigrin... more A series of simple aza and diaza bicyclic quinones related to the AB ring system of streptonigrin (1) have been synthesized and tested in vitro for their ability to degrade DNA under conditions similar to those used with the parent drug. The results obtained from a study of 22 quinones indicate that there is a quantitative linear relationship between their reduction potentials and the rate at which they degrade DNA under identical conditions in vitro. Almost all of the synthetic substances were superior to 1 in their DNA-degrading ability.
Molecular & cellular proteomics : MCP, 2006
Apurinic/apyrimidinic (AP) sites, a prominent type of DNA damage, are repaired through the base e... more Apurinic/apyrimidinic (AP) sites, a prominent type of DNA damage, are repaired through the base excision repair mechanism in both prokaryotes and eukaryotes and may interfere with many other cellular processes. A full repertoire of AP site-binding proteins in cells is presently unknown, preventing reliable assessment of harm inflicted by these ubiquitous lesions and of their involvement in the flux of DNA metabolism. We present a proteomics-based strategy for assembling at least a partial catalogue of proteins capable of binding AP sites in DNA. The general scheme relies on the sensitivity of many AP site-bound protein species to NaBH(4) cross-linking. An affinity-tagged substrate is used to facilitate isolation of the cross-linked species, which are then separated and analyzed by mass spectrometry methods. We report identification of seven proteins from Escherichia coli (AroF, DnaK, MutM, PolA, TnaA, TufA, and UvrA) and two proteins from bakers' yeast (ARC1 and Ygl245wp) react...
Biochemistry, 1996
The MutY protein of Escherichia coli removes mismatched deoxyadenine residues from DNA. In this s... more The MutY protein of Escherichia coli removes mismatched deoxyadenine residues from DNA. In this study, duplex oligodeoxynucleotides containing modified bases are used as model substrates for this enzyme. In contrast to a recent report [Lu, A.-L., et al. (1995) J. Biol. Chem. 270, 23582], dA:8oxo-dG appears to be the preferred natural substrate for MutY, as evidenced by the specificity constants (k cat /K m) for dA:8-oxo-dG and dA:dG of 39 600 × 10-6 and 383 × 10-6 (min-1 nM-1), respectively. k cat for the duplex containing dA:dG was highest at lower pH; the rate of cleavage for the duplex containing dA:8-oxo-dG was unaffected over a pH range of 5.5-8.0. The presence of an 8-oxo function in dG increased significantly the rate of removal of dA from all substrates tested. Replacement of dA by rA reduced the specificity constant of dA:8-oxo-dG to 294 × 10-6 (min-1 nM-1), whereas replacement of dA by 2′-O-methyladenosine virtually abolished enzymatic activity. Modifications of the dG moiety generally were better tolerated than those of dA; however, introduction of a methyl ether at the 6 position of dG produced a noncleavable substrate and replacement of dG by 2′-O-methylguanosine generated a substrate with a low specificity constant. Rates of cleavage of duplexes containing dA:dC and dA:tetrahydrofuran were three orders of magnitude lower than the reference substrate. Duplexes containing a carbocyclic analog of dA were not cleaved. A model is proposed to explain the recognition of DNA substrates by MutY and the catalytic properties of this enzyme.
Biochemistry, 2004
Codon 273 ((5)(')CGT) of the human P53 gene is a mutational hot spot for the environmental c... more Codon 273 ((5)(')CGT) of the human P53 gene is a mutational hot spot for the environmental carcinogen benzo[a]pyrene. We incorporated a single (+)- or (-)-trans-anti-benzo[a]pyrene diol epoxide (BPDE) DNA adduct at the second position of codon 273 of the human P53 gene and explored the mutagenic potential of this lesion in mammalian cells. Oligodeoxyribonucleotides ((5)(')GAGGTGCG(BPDE)TGTTTGT) modified with (+)- or (-)-trans-dG-N(2)-BPDE were incorporated into single-stranded shuttle vectors and transfected into simian kidney cells. Progeny plasmids were then used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and spectrum produced by the adducted base. We determined the mutational frequencies associated with (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE adduction to be 26.5% and 17.5%, respectively. The predominant mutations generated by both stereoisomers were G --> T transversions, with some G --> A transitions. When the cytosine 5' to dG-N(2)-BPDE was replaced by 5-methylcytosine, the mutational frequencies of (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE were reduced to 11.1% and 10.6%, respectively, while the mutational specificity remained unchanged. Thus, the mutational "hot spot" at codon 273 in P53 may reflect either sequence-specific reactivity of BPDE and/or inefficient repair of BPDE-DNA adducts positioned at this site.
Biochemistry, 1998
The abasic site in DNA may arise spontaneously, as a result of nucleotide base damage, or as an i... more The abasic site in DNA may arise spontaneously, as a result of nucleotide base damage, or as an intermediate in glycosylase-mediated DNA-repair pathways. It is the most common damage found in DNA. We have examined the consequences of this lesion and its sequence context on DNA duplex structure, as well as the thermal and thermodynamic stability of the duplex, including the energetic origins of that stability. To this end, we incorporated a tetrahydrofuran abasic site analogue into a family of 13-mer DNA duplexes, wherein the base opposite the lesion (A, C, G, or T) and the base pairs neighboring the lesion (C‚G or G‚C) were systematically varied and characterized by a combination of spectroscopic and calorimetric techniques. The resulting data allowed us to reach the following conclusions: (i) the presence of the lesion in all sequence contexts studied does not alter the global B-form conformation characteristic of the parent undamaged duplex; (ii) the presence of the lesion induces a significant enthalpic destabilization of the duplex, with the magnitude of this effect being dependent on the sequence context; (iii) the thermodynamic impact of the lesion is dominated by the identity of the neighboring base pairs, with the cross strand partner base exerting only a secondary thermodynamic effect on duplex properties. In the aggregate, our data reveal that even in the absence of lesion-induced alterations in global structure, the abasic lesion can significantly alter the thermodynamic properties of the host duplex, with the magntiude of this impact being strongly dependent on sequence context.
Biochemistry, 1997
Vinyl chloride reacts with cellular DNA producing 3,N 4 -etheno-2′-deoxycytidine ( C) along with ... more Vinyl chloride reacts with cellular DNA producing 3,N 4 -etheno-2′-deoxycytidine ( C) along with other exocyclic adducts. The solution structure of an oligodeoxynucleotide duplex containing an C‚dG base pair was determined by high-resolution NMR spectroscopy and molecular dynamics simulations. NMR data indicated that the duplex adopts a right-handed helical structure having all residues in anti orientation around the glycosylic torsion angle. The C adduct has a sugar pucker in the C3′endo/C4′-exo region while the rest of the residues are in the C2′-endo/C3′-exo range. NOE interactions established Watson-Crick alignments for canonical base pairs of the duplex. The imino proton of the lesion-containing base pair resonated as a sharp signal that was resistant to water exchange, suggesting hydrogen bonding. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. The refined structures are slightly bent at the lesion site without major perturbations of the sugar-phosphate backbone. The adduct is displaced and shifted toward the major groove of the helix while its partner on the complementary strand remains stacked. The C-(anti)‚dG(anti) base pair alignment is sheared and stabilized by the formation of hydrogen bonds. The biological implications of structures of C-containing DNA duplexes are discussed.
Biochemistry, 1995
As part of an overall program to characterize the impact of mutagenic lesions on the physiochemic... more As part of an overall program to characterize the impact of mutagenic lesions on the physiochemical properties of DNA, we report here the results of a comparative spectroscopic and calorimetric study on a family of DNA duplexes both with and without the oxidative lesion 2'-deoxy-7
Biochemistry, 1991
Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opp... more Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opposite an abasic site in the center of a D N A oligomer duplex. N M R and energy minimization studies were performed on the 1 ,M-propanodeoxyguanosine exocyclic adduct (X) positioned opposite a tetrahydrofuran abasic site (F) with the dual lesions located in the center of the (Cl-A2-T3-G4-XS-G6-T7-A8-C9)-(GlO-T1 l-A12-C13-F14-C15-A16-T17-G18) X-F 9-mer duplex. Two-dimensional N M R experiments establish that the X-F 9-mer helix is right-handed with Watson-Crick A.T and G C base pairing on either side of the lesion site. NOES are detected from the methylene protons of the exocyclic ring of X5 to the imino protons of G 4 C 1 5 and G 6 C 1 3 which flank the lesion site, as well as to the H1' and H1" protons of the cross strand F14 tetrahydrofuran moiety. These N M R results establish that the exocyclic adduct X5 is positioned between flanking G 4 C 1 5 and G 6 C 1 3 base pairs and directed toward the abasic lesion F14 on the partner strand. These studies establish that the exocyclic ring of the 1,M-propanodeoxyguanosine adduct fits into the cavity generated by the abasic site.
Biochemistry, 2000
To understand how the active site of a DNA polymerase might modulate the coding of 8-oxo-7,8-dihy... more To understand how the active site of a DNA polymerase might modulate the coding of 8-oxo-7,8-dihydrodeoxyguanine (8-oxodG), we performed steady-state kinetic analyses using wild-type DNA polymerase (pol ) and two active-site mutants. We compared the coding of these polymerases by calculating the ratio of efficiencies for incorporation of dATP and dCTP opposite 8-oxodG and for incorporation of 8-oxodGTP opposite dA and dC. For wild-type pol , there is a 2:1 preference for incorporation of dCTP over dATP opposite 8-oxodG using a 5′-phosphorylated 4-base gap substrate. Mutation of either Asn279 or Arg283 to alanine has almost no effect on the ratio. 8-OxodGTP is preferentially incorporated opposite a template dA (24:1) by wild-type pol ; mutation of Asn279 to alanine results dramatic change whereby there is preferential incorporation of 8-oxodGTP opposite dC (14:1). This suggests that interactions of 8-oxodGTP with Asn279 in the polymerase active site may alter the conformation of 8-oxodGTP and therefore alter its misincorporation. .
Biochemistry, 1993
This study was designed to establish the miscoding potential of 8-oxo-7,8-dihydrodeoxyadenosine (... more This study was designed to establish the miscoding potential of 8-oxo-7,8-dihydrodeoxyadenosine (8-oxo-dA). Oligodeoxynucleotides modified site-specifically with 8-oxo-dA were used as templates in primer extension reactions catalyzed by DNA polymerase I (Klenow fragment), DNA polymerase alpha (pol alpha), or DNA polymerase beta (pol beta). dTMP or dGMP is incorporated opposite 8-oxo-dA when either of these dNTPs is provided as substrate for DNA polymerase. dTMP is incorporated exclusively opposite 8-oxo-dA when all four dNTPs are present in the reaction mixture at equimolar concentrations. Chain extension is catalyzed efficiently by Klenow fragment and pol beta under conditions where 8-oxo-dA is paired with dT at the 3' terminus of the primed DNA template. Chain extension catalyzed by pol alpha proceeds more slowly. As shown by steady-state kinetic experiments, incorporation of dGMP is higher in reactions catalyzed by pol beta than by Klenow fragment or pol alpha. The dG-8-oxo-dA pair is extended efficiently from the 3' terminus in the absence of dTTP. We conclude that DNA containing 8-oxo-dA is capable of miscoding; however, unlike 8-oxo-dG, the mutagenic potential of this lesion is limited.
Biochemistry, 1991
Proton NMR studies are reported on the complementary d(C1-C2-A3-C4-T5-A6-oxo-G7-T8-C9-A10-C11-C12... more Proton NMR studies are reported on the complementary d(C1-C2-A3-C4-T5-A6-oxo-G7-T8-C9-A10-C11-C12).d(G13-G14-T15- G16-A17-A18-T19- A20-G21-T22-G23-G24) dodecanucleotide duplex (designated 8-oxo-7H-dG.dA 12-mer), which contains a centrally located 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) residue, a group commonly found in DNA that has been exposed to ionizing radiation or oxidizing free radicals. From the NMR spectra it can be deduced that this moiety exists as two tautomers, or gives rise to two DNA conformations, that are in equilibrium and that exchange slowly. The present study focuses on the major component of the equilibrium that originates in the 6,8-dioxo tautomer of 8-oxo-7H-dG. We have assigned the exchangeable NH1, NH7, and NH2-2 base protons located on the Watson-Crick and Hoogsteen edges of 8-oxo-7H-dG7 in the 8-oxo-7H-dG.dA 12-mer duplex, using an analysis of one- and two-dimensional nuclear Overhauser enhancement (NOE) data in H2O solution. The observed NOEs derived from the NH7 proton of 8-oxo-7H-dG7 to the H2 and NH2-6 protons of dA18 establish an 8-oxo-7H-dG7(syn).dA 18(anti) alignment at the lesion site in the 8-oxo-7H-dG.dA 12-mer duplex in solution. This alignment, which places the 8-oxo group in the minor groove, was further characterized by an analysis of the NOESY spectrum of the 8-oxo-7H-dG.dA 12-mer duplex in D2O solution. We were able to detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(A6-oxo-G7-T8).d(A17-A18-T19) trinucleotide segment centered about the lesion site that establishes stacking of the oxo-dG7(syn).dA(anti) pair between stable Watson-Crick dA6.dT19 and dT8.dA17 base pairs with minimal perturbation of the helix. Thus, both strands of the 8-oxo-7H-dG.dA 12-mer duplex adopt right-handed conformations at and adjacent to the lesion site, the unmodified bases adopt anti glycosidic torsion angles, and the bases are stacked into the helix. The energy-minimized conformation of the central d(A6-oxo-G7-T8).d(A17-A18-T19) segment requires that the 8-oxo-7H-dG7(syn).dA18(anti) alignment be stabilized by two hydrogen bonds from NH7 and O6 of 8-oxo-7H-dG7(syn) to N1 and NH2-6 of dA18(anti), respectively, at the lesion site.(ABSTRACT TRUNCATED AT 400 WORDS)
Advances in Molecular Toxicology
Biopolymers, 2015
The magnitude and nature of lesion-induced energetic perturbations empirically correlate with mut... more The magnitude and nature of lesion-induced energetic perturbations empirically correlate with mutagenicity/cytotoxicity profiles and can be predictive of lesion outcomes during polymerase-mediated replication in vitro. In this study, we assess the sequence and counterbase-dependent energetic impact of the Thymine glycol (Tg ) lesion on a family of deoxyoligonucleotide duplexes. Tg damage arises from thymine and methyl-cytosine exposure to oxidizing agents or radiation-generated free-radicals. The Tg lesion blocks polymerase-mediated DNA replication in vitro and the unrepaired site elicits cytotoxic lethal consequences in vivo. Our combined calorimetric and spectroscopic characterization correlates Tg -induced energetic perturbations with biological and structural properties. Specifically, we incorporate a 5R-Tg isomer centered within the tridecanucleotide sequence 5'-GCGTACXCATGCG-3' (X = Tg or T) which is hybridized with the corresponding complementary sequence 5'-CGCATGNGTACGC-3' (N = A, G, T, C) to generate families of Tg -damaged (Tg ·N) and lesion-free (T·N) duplexes. We demonstrate that the magnitude and nature of the Tg destabilizing impact is dependent on counterbase identity (i.e., A ∼ G < T < C). The observation that a Tg lesion is less destabilizing when positioned opposite purines suggests that favorable counterbase stacking interactions may partially compensate lesion-induced perturbations. Moreover, the destabilizing energies of Tg ·N duplexes parallel their respective lesion-free T·N mismatch counterparts (i.e., G < T < C). Elucidation of Tg -induced destabilization relative to the corresponding undamaged mismatch energetics allows resolution of lesion-specific and sequence-dependent impacts. The Tg -induced energetic perturbations are consistent with its replication blocking properties and may serve as differential recognition elements for discrimination by the cellular repair machinery. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 491-508, 2015.
Archives of toxicology, 2015
Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and... more Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adducts formation, is well documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. To better elucidate some aspects of this process, we studied cell cycle distribution and cell survival of renal epithelial cells treated with AAI at low and high doses. A low dose of AA induces cell cycle arrest in G2/M phase via activation of DNA damage checkpoint pathway ATM-Chk2-p53-p21. DNA damage signaling pathway is activated more likely via increased production of reactive oxygen species (ROS) caused by AA treatment then via DNA damage induced directly by AA. Higher AA concentration induced cell death partly via apoptosis. Since mitogen-activated protein kinases play an important role in cell survival, death and cell cycle progression, we assayed their...
Nucleic acids research, 2015
Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal... more Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that ...
Clinical Journal of the American Society of Nephrology, 2015
Improvements in agricultural practices in Croatia have reduced exposure to consumption of aristol... more Improvements in agricultural practices in Croatia have reduced exposure to consumption of aristolochic acid-contaminated flour and development of endemic (Balkan) nephropathy. Therefore, it was hypothesized that Bosnian immigrants who settled in an endemic area in Croatia 15-30 years ago would be at lower risk of developing endemic nephropathy because of reduced exposure to aristolochic acid. To test this hypothesis, past and present exposure to aristolochic acid, proximal tubule damage as a hallmark of endemic nephropathy, and prevalence of CKD in Bosnian immigrants were analyzed. In this cross-sectional observational study from 2005 to 2010, 2161 farmers were divided into groups: indigenous inhabitants from endemic nephropathy and nonendemic nephropathy villages and Bosnian immigrants; α-1 microglobulin-to-creatinine ratio >31.5 mg/g and eGFR<60 ml/min per 1.73 m(2) were considered to be abnormal. CKD and proximal tubule damage prevalence was significantly lower in Bosnian immigrants than inhabitants of endemic nephropathy villages (6.9% versus 16.6%; P<0.001; 1.3% versus 7.3%; P=0.003, respectively); 20 years ago, Bosnian immigrants observed fewer Aristolochia clematitis in cultivated fields (41.9% versus 67.8%) and fewer seeds among wheat seeds (6.1% versus 35.6%) and ate more purchased than homemade bread compared with Croatian farmers from endemic nephropathy villages (38.5% versus 14.8%, P<0.001). Both Croatian farmers and Bosnian immigrants observe significantly fewer Aristolochia plants growing in their fields compared with 15-30 years ago. Prior aristolochic acid exposure was associated with proximal tubule damage (odds ratio, 1.64; 95% confidence interval, 1.04 to 2.58; P=0.02), whereas present exposure was not (odds ratio, 1.31; 95% confidence interval, 0.75 to 2.30; P=0.33). Furthermore, immigrant status was an independent negative predictor of proximal tubule damage (odds ratio, 0.40; 95% confidence interval, 0.19 to 0.86; P=0.02). Bosnian immigrants and autochthonous Croats residing in endemic areas are exposed significantly less to ingestion of aristolochic acid than in the past. The prevalence of endemic nephropathy and its associated urothelial cancers is predicted to decrease over time.
Nephrology Dialysis Transplantation, 2013
Urologic Oncology: Seminars and Original Investigations, 2014
Upper tract urothelial carcinoma (UTUC) is a rare disease in Western countries and garners little... more Upper tract urothelial carcinoma (UTUC) is a rare disease in Western countries and garners little focused attention in urologic and oncologic circles. We report highlights from the first symposium on UTUC. All participants were asked to provide a summary of their presentation to be included as part of these proceedings. Submitted summaries were synthesized into this document. All contributors reviewed and provided input on the final draft. Five highlights are included in this report, including landmark research that not only reveals the likely cause of Balkan endemic nephropathy and associated UTUC but also links it directly to UTUC in Taiwan. Because of the ubiquitous use of Aristolochia plants in these herbal remedies, a public health problem of considerable magnitude is anticipated in Asian countries. Gene expression signatures reveal some differential expression in bladder carcinoma, such as CLCA2 and GABRE. Few urinary markers have proven utility for the diagnosis and follow-up of UTUC, and no tissue or blood-based markers are currently undergoing clinical application. Novel endoscopic therapies provide some hope of improving tissue sampling, diagnosis, and kidney-sparing therapeutics, but the greatest potential lies in improving clinical (preoperative) risk stratification, which is critically limited in this disease. Biomarkers, currently untested, hold promise in identifying patients most likely to benefit from perioperative chemotherapy and at high risk from cisplatin-induced nephrotoxicity. Despite its rarity in the West, UTUC is reaching potentially epidemic proportions in the East because of exposure to carcinogenic herbal remedies. Critical trials are needed to improve our understanding and treatment of UTUC. Because of the broad range of comorbid conditions in patients suffering from this disease, it is the consensus of the participants that future clinical trials should be practical in design and applicable to a broad range of patients, diverging from the current dogma of narrow patient selection criteria in clinical trials. Practical designs would maximize accrual for a still uncommon disease, and their findings would be applicable to a larger proportion of patients than current narrowly selected designs.
Mutation Research/Reviews in Mutation Research, 2013
Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental car... more Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental carcinogens. Here we review the pieces of evidence that support the role of aristolochic acid (AA) in inducing a mutational fingerprint in the tumor suppressor gene TP53 in urothelial carcinomas of the upper urinary tract (UUT). Exposure to AA, a nitrophenathrene carboxylic acid present in certain herbal remedies and in flour prepared from wheat grain contaminated with seeds of Aristolochia clematitis, has been linked to chronic nephropathy and UUT. TP53 mutations in UUT of individuals exposed to AA reveal a unique pattern of mutations characterized by A to T transversions on the non-transcribed strand, which cluster at hotspots rarely mutated in other cancers. This unusual pattern, originally discovered in UUTs from two different populations, one in Taiwan, and one in the Balkans, has been reproduced experimentally by treating mouse cells that harbor human TP53 sequences with AA. The convergence of molecular epidemiological and experimental data establishes a clear causal association between exposure to the human carcinogen AA and UUT. Despite bans on the sale of herbs containing AA, their use continues, raising global public health concern and an urgent need to identify populations at risk.
Journal of Molecular Modeling, 1999
DNA is a long, thread-like macromolecule composed of two helical polynucleotide chains. The chain... more DNA is a long, thread-like macromolecule composed of two helical polynucleotide chains. The chains are coiled around a common axis and are antisymmetric with respect to the helical axis. The most frequent conformation is a so-called B-DNA in which the diameter of the double helix is approximately 20 Å.
Journal of Medicinal Chemistry, 1986
A series of simple aza and diaza bicyclic quinones related to the AB ring system of streptonigrin... more A series of simple aza and diaza bicyclic quinones related to the AB ring system of streptonigrin (1) have been synthesized and tested in vitro for their ability to degrade DNA under conditions similar to those used with the parent drug. The results obtained from a study of 22 quinones indicate that there is a quantitative linear relationship between their reduction potentials and the rate at which they degrade DNA under identical conditions in vitro. Almost all of the synthetic substances were superior to 1 in their DNA-degrading ability.
Molecular & cellular proteomics : MCP, 2006
Apurinic/apyrimidinic (AP) sites, a prominent type of DNA damage, are repaired through the base e... more Apurinic/apyrimidinic (AP) sites, a prominent type of DNA damage, are repaired through the base excision repair mechanism in both prokaryotes and eukaryotes and may interfere with many other cellular processes. A full repertoire of AP site-binding proteins in cells is presently unknown, preventing reliable assessment of harm inflicted by these ubiquitous lesions and of their involvement in the flux of DNA metabolism. We present a proteomics-based strategy for assembling at least a partial catalogue of proteins capable of binding AP sites in DNA. The general scheme relies on the sensitivity of many AP site-bound protein species to NaBH(4) cross-linking. An affinity-tagged substrate is used to facilitate isolation of the cross-linked species, which are then separated and analyzed by mass spectrometry methods. We report identification of seven proteins from Escherichia coli (AroF, DnaK, MutM, PolA, TnaA, TufA, and UvrA) and two proteins from bakers' yeast (ARC1 and Ygl245wp) react...
Biochemistry, 1996
The MutY protein of Escherichia coli removes mismatched deoxyadenine residues from DNA. In this s... more The MutY protein of Escherichia coli removes mismatched deoxyadenine residues from DNA. In this study, duplex oligodeoxynucleotides containing modified bases are used as model substrates for this enzyme. In contrast to a recent report [Lu, A.-L., et al. (1995) J. Biol. Chem. 270, 23582], dA:8oxo-dG appears to be the preferred natural substrate for MutY, as evidenced by the specificity constants (k cat /K m) for dA:8-oxo-dG and dA:dG of 39 600 × 10-6 and 383 × 10-6 (min-1 nM-1), respectively. k cat for the duplex containing dA:dG was highest at lower pH; the rate of cleavage for the duplex containing dA:8-oxo-dG was unaffected over a pH range of 5.5-8.0. The presence of an 8-oxo function in dG increased significantly the rate of removal of dA from all substrates tested. Replacement of dA by rA reduced the specificity constant of dA:8-oxo-dG to 294 × 10-6 (min-1 nM-1), whereas replacement of dA by 2′-O-methyladenosine virtually abolished enzymatic activity. Modifications of the dG moiety generally were better tolerated than those of dA; however, introduction of a methyl ether at the 6 position of dG produced a noncleavable substrate and replacement of dG by 2′-O-methylguanosine generated a substrate with a low specificity constant. Rates of cleavage of duplexes containing dA:dC and dA:tetrahydrofuran were three orders of magnitude lower than the reference substrate. Duplexes containing a carbocyclic analog of dA were not cleaved. A model is proposed to explain the recognition of DNA substrates by MutY and the catalytic properties of this enzyme.
Biochemistry, 2004
Codon 273 ((5)(')CGT) of the human P53 gene is a mutational hot spot for the environmental c... more Codon 273 ((5)(')CGT) of the human P53 gene is a mutational hot spot for the environmental carcinogen benzo[a]pyrene. We incorporated a single (+)- or (-)-trans-anti-benzo[a]pyrene diol epoxide (BPDE) DNA adduct at the second position of codon 273 of the human P53 gene and explored the mutagenic potential of this lesion in mammalian cells. Oligodeoxyribonucleotides ((5)(')GAGGTGCG(BPDE)TGTTTGT) modified with (+)- or (-)-trans-dG-N(2)-BPDE were incorporated into single-stranded shuttle vectors and transfected into simian kidney cells. Progeny plasmids were then used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and spectrum produced by the adducted base. We determined the mutational frequencies associated with (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE adduction to be 26.5% and 17.5%, respectively. The predominant mutations generated by both stereoisomers were G --> T transversions, with some G --> A transitions. When the cytosine 5' to dG-N(2)-BPDE was replaced by 5-methylcytosine, the mutational frequencies of (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE were reduced to 11.1% and 10.6%, respectively, while the mutational specificity remained unchanged. Thus, the mutational "hot spot" at codon 273 in P53 may reflect either sequence-specific reactivity of BPDE and/or inefficient repair of BPDE-DNA adducts positioned at this site.
Biochemistry, 1998
The abasic site in DNA may arise spontaneously, as a result of nucleotide base damage, or as an i... more The abasic site in DNA may arise spontaneously, as a result of nucleotide base damage, or as an intermediate in glycosylase-mediated DNA-repair pathways. It is the most common damage found in DNA. We have examined the consequences of this lesion and its sequence context on DNA duplex structure, as well as the thermal and thermodynamic stability of the duplex, including the energetic origins of that stability. To this end, we incorporated a tetrahydrofuran abasic site analogue into a family of 13-mer DNA duplexes, wherein the base opposite the lesion (A, C, G, or T) and the base pairs neighboring the lesion (C‚G or G‚C) were systematically varied and characterized by a combination of spectroscopic and calorimetric techniques. The resulting data allowed us to reach the following conclusions: (i) the presence of the lesion in all sequence contexts studied does not alter the global B-form conformation characteristic of the parent undamaged duplex; (ii) the presence of the lesion induces a significant enthalpic destabilization of the duplex, with the magnitude of this effect being dependent on the sequence context; (iii) the thermodynamic impact of the lesion is dominated by the identity of the neighboring base pairs, with the cross strand partner base exerting only a secondary thermodynamic effect on duplex properties. In the aggregate, our data reveal that even in the absence of lesion-induced alterations in global structure, the abasic lesion can significantly alter the thermodynamic properties of the host duplex, with the magntiude of this impact being strongly dependent on sequence context.
Biochemistry, 1997
Vinyl chloride reacts with cellular DNA producing 3,N 4 -etheno-2′-deoxycytidine ( C) along with ... more Vinyl chloride reacts with cellular DNA producing 3,N 4 -etheno-2′-deoxycytidine ( C) along with other exocyclic adducts. The solution structure of an oligodeoxynucleotide duplex containing an C‚dG base pair was determined by high-resolution NMR spectroscopy and molecular dynamics simulations. NMR data indicated that the duplex adopts a right-handed helical structure having all residues in anti orientation around the glycosylic torsion angle. The C adduct has a sugar pucker in the C3′endo/C4′-exo region while the rest of the residues are in the C2′-endo/C3′-exo range. NOE interactions established Watson-Crick alignments for canonical base pairs of the duplex. The imino proton of the lesion-containing base pair resonated as a sharp signal that was resistant to water exchange, suggesting hydrogen bonding. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. The refined structures are slightly bent at the lesion site without major perturbations of the sugar-phosphate backbone. The adduct is displaced and shifted toward the major groove of the helix while its partner on the complementary strand remains stacked. The C-(anti)‚dG(anti) base pair alignment is sheared and stabilized by the formation of hydrogen bonds. The biological implications of structures of C-containing DNA duplexes are discussed.
Biochemistry, 1995
As part of an overall program to characterize the impact of mutagenic lesions on the physiochemic... more As part of an overall program to characterize the impact of mutagenic lesions on the physiochemical properties of DNA, we report here the results of a comparative spectroscopic and calorimetric study on a family of DNA duplexes both with and without the oxidative lesion 2'-deoxy-7
Biochemistry, 1991
Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opp... more Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opposite an abasic site in the center of a D N A oligomer duplex. N M R and energy minimization studies were performed on the 1 ,M-propanodeoxyguanosine exocyclic adduct (X) positioned opposite a tetrahydrofuran abasic site (F) with the dual lesions located in the center of the (Cl-A2-T3-G4-XS-G6-T7-A8-C9)-(GlO-T1 l-A12-C13-F14-C15-A16-T17-G18) X-F 9-mer duplex. Two-dimensional N M R experiments establish that the X-F 9-mer helix is right-handed with Watson-Crick A.T and G C base pairing on either side of the lesion site. NOES are detected from the methylene protons of the exocyclic ring of X5 to the imino protons of G 4 C 1 5 and G 6 C 1 3 which flank the lesion site, as well as to the H1' and H1" protons of the cross strand F14 tetrahydrofuran moiety. These N M R results establish that the exocyclic adduct X5 is positioned between flanking G 4 C 1 5 and G 6 C 1 3 base pairs and directed toward the abasic lesion F14 on the partner strand. These studies establish that the exocyclic ring of the 1,M-propanodeoxyguanosine adduct fits into the cavity generated by the abasic site.
Biochemistry, 2000
To understand how the active site of a DNA polymerase might modulate the coding of 8-oxo-7,8-dihy... more To understand how the active site of a DNA polymerase might modulate the coding of 8-oxo-7,8-dihydrodeoxyguanine (8-oxodG), we performed steady-state kinetic analyses using wild-type DNA polymerase (pol ) and two active-site mutants. We compared the coding of these polymerases by calculating the ratio of efficiencies for incorporation of dATP and dCTP opposite 8-oxodG and for incorporation of 8-oxodGTP opposite dA and dC. For wild-type pol , there is a 2:1 preference for incorporation of dCTP over dATP opposite 8-oxodG using a 5′-phosphorylated 4-base gap substrate. Mutation of either Asn279 or Arg283 to alanine has almost no effect on the ratio. 8-OxodGTP is preferentially incorporated opposite a template dA (24:1) by wild-type pol ; mutation of Asn279 to alanine results dramatic change whereby there is preferential incorporation of 8-oxodGTP opposite dC (14:1). This suggests that interactions of 8-oxodGTP with Asn279 in the polymerase active site may alter the conformation of 8-oxodGTP and therefore alter its misincorporation. .
Biochemistry, 1993
This study was designed to establish the miscoding potential of 8-oxo-7,8-dihydrodeoxyadenosine (... more This study was designed to establish the miscoding potential of 8-oxo-7,8-dihydrodeoxyadenosine (8-oxo-dA). Oligodeoxynucleotides modified site-specifically with 8-oxo-dA were used as templates in primer extension reactions catalyzed by DNA polymerase I (Klenow fragment), DNA polymerase alpha (pol alpha), or DNA polymerase beta (pol beta). dTMP or dGMP is incorporated opposite 8-oxo-dA when either of these dNTPs is provided as substrate for DNA polymerase. dTMP is incorporated exclusively opposite 8-oxo-dA when all four dNTPs are present in the reaction mixture at equimolar concentrations. Chain extension is catalyzed efficiently by Klenow fragment and pol beta under conditions where 8-oxo-dA is paired with dT at the 3' terminus of the primed DNA template. Chain extension catalyzed by pol alpha proceeds more slowly. As shown by steady-state kinetic experiments, incorporation of dGMP is higher in reactions catalyzed by pol beta than by Klenow fragment or pol alpha. The dG-8-oxo-dA pair is extended efficiently from the 3' terminus in the absence of dTTP. We conclude that DNA containing 8-oxo-dA is capable of miscoding; however, unlike 8-oxo-dG, the mutagenic potential of this lesion is limited.
Biochemistry, 1991
Proton NMR studies are reported on the complementary d(C1-C2-A3-C4-T5-A6-oxo-G7-T8-C9-A10-C11-C12... more Proton NMR studies are reported on the complementary d(C1-C2-A3-C4-T5-A6-oxo-G7-T8-C9-A10-C11-C12).d(G13-G14-T15- G16-A17-A18-T19- A20-G21-T22-G23-G24) dodecanucleotide duplex (designated 8-oxo-7H-dG.dA 12-mer), which contains a centrally located 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) residue, a group commonly found in DNA that has been exposed to ionizing radiation or oxidizing free radicals. From the NMR spectra it can be deduced that this moiety exists as two tautomers, or gives rise to two DNA conformations, that are in equilibrium and that exchange slowly. The present study focuses on the major component of the equilibrium that originates in the 6,8-dioxo tautomer of 8-oxo-7H-dG. We have assigned the exchangeable NH1, NH7, and NH2-2 base protons located on the Watson-Crick and Hoogsteen edges of 8-oxo-7H-dG7 in the 8-oxo-7H-dG.dA 12-mer duplex, using an analysis of one- and two-dimensional nuclear Overhauser enhancement (NOE) data in H2O solution. The observed NOEs derived from the NH7 proton of 8-oxo-7H-dG7 to the H2 and NH2-6 protons of dA18 establish an 8-oxo-7H-dG7(syn).dA 18(anti) alignment at the lesion site in the 8-oxo-7H-dG.dA 12-mer duplex in solution. This alignment, which places the 8-oxo group in the minor groove, was further characterized by an analysis of the NOESY spectrum of the 8-oxo-7H-dG.dA 12-mer duplex in D2O solution. We were able to detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(A6-oxo-G7-T8).d(A17-A18-T19) trinucleotide segment centered about the lesion site that establishes stacking of the oxo-dG7(syn).dA(anti) pair between stable Watson-Crick dA6.dT19 and dT8.dA17 base pairs with minimal perturbation of the helix. Thus, both strands of the 8-oxo-7H-dG.dA 12-mer duplex adopt right-handed conformations at and adjacent to the lesion site, the unmodified bases adopt anti glycosidic torsion angles, and the bases are stacked into the helix. The energy-minimized conformation of the central d(A6-oxo-G7-T8).d(A17-A18-T19) segment requires that the 8-oxo-7H-dG7(syn).dA18(anti) alignment be stabilized by two hydrogen bonds from NH7 and O6 of 8-oxo-7H-dG7(syn) to N1 and NH2-6 of dA18(anti), respectively, at the lesion site.(ABSTRACT TRUNCATED AT 400 WORDS)
Advances in Molecular Toxicology
Biopolymers, 2015
The magnitude and nature of lesion-induced energetic perturbations empirically correlate with mut... more The magnitude and nature of lesion-induced energetic perturbations empirically correlate with mutagenicity/cytotoxicity profiles and can be predictive of lesion outcomes during polymerase-mediated replication in vitro. In this study, we assess the sequence and counterbase-dependent energetic impact of the Thymine glycol (Tg ) lesion on a family of deoxyoligonucleotide duplexes. Tg damage arises from thymine and methyl-cytosine exposure to oxidizing agents or radiation-generated free-radicals. The Tg lesion blocks polymerase-mediated DNA replication in vitro and the unrepaired site elicits cytotoxic lethal consequences in vivo. Our combined calorimetric and spectroscopic characterization correlates Tg -induced energetic perturbations with biological and structural properties. Specifically, we incorporate a 5R-Tg isomer centered within the tridecanucleotide sequence 5'-GCGTACXCATGCG-3' (X = Tg or T) which is hybridized with the corresponding complementary sequence 5'-CGCATGNGTACGC-3' (N = A, G, T, C) to generate families of Tg -damaged (Tg ·N) and lesion-free (T·N) duplexes. We demonstrate that the magnitude and nature of the Tg destabilizing impact is dependent on counterbase identity (i.e., A ∼ G < T < C). The observation that a Tg lesion is less destabilizing when positioned opposite purines suggests that favorable counterbase stacking interactions may partially compensate lesion-induced perturbations. Moreover, the destabilizing energies of Tg ·N duplexes parallel their respective lesion-free T·N mismatch counterparts (i.e., G < T < C). Elucidation of Tg -induced destabilization relative to the corresponding undamaged mismatch energetics allows resolution of lesion-specific and sequence-dependent impacts. The Tg -induced energetic perturbations are consistent with its replication blocking properties and may serve as differential recognition elements for discrimination by the cellular repair machinery. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 491-508, 2015.
Archives of toxicology, 2015
Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and... more Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adducts formation, is well documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. To better elucidate some aspects of this process, we studied cell cycle distribution and cell survival of renal epithelial cells treated with AAI at low and high doses. A low dose of AA induces cell cycle arrest in G2/M phase via activation of DNA damage checkpoint pathway ATM-Chk2-p53-p21. DNA damage signaling pathway is activated more likely via increased production of reactive oxygen species (ROS) caused by AA treatment then via DNA damage induced directly by AA. Higher AA concentration induced cell death partly via apoptosis. Since mitogen-activated protein kinases play an important role in cell survival, death and cell cycle progression, we assayed their...
Nucleic acids research, 2015
Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal... more Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that ...
Clinical Journal of the American Society of Nephrology, 2015
Improvements in agricultural practices in Croatia have reduced exposure to consumption of aristol... more Improvements in agricultural practices in Croatia have reduced exposure to consumption of aristolochic acid-contaminated flour and development of endemic (Balkan) nephropathy. Therefore, it was hypothesized that Bosnian immigrants who settled in an endemic area in Croatia 15-30 years ago would be at lower risk of developing endemic nephropathy because of reduced exposure to aristolochic acid. To test this hypothesis, past and present exposure to aristolochic acid, proximal tubule damage as a hallmark of endemic nephropathy, and prevalence of CKD in Bosnian immigrants were analyzed. In this cross-sectional observational study from 2005 to 2010, 2161 farmers were divided into groups: indigenous inhabitants from endemic nephropathy and nonendemic nephropathy villages and Bosnian immigrants; α-1 microglobulin-to-creatinine ratio >31.5 mg/g and eGFR<60 ml/min per 1.73 m(2) were considered to be abnormal. CKD and proximal tubule damage prevalence was significantly lower in Bosnian immigrants than inhabitants of endemic nephropathy villages (6.9% versus 16.6%; P<0.001; 1.3% versus 7.3%; P=0.003, respectively); 20 years ago, Bosnian immigrants observed fewer Aristolochia clematitis in cultivated fields (41.9% versus 67.8%) and fewer seeds among wheat seeds (6.1% versus 35.6%) and ate more purchased than homemade bread compared with Croatian farmers from endemic nephropathy villages (38.5% versus 14.8%, P<0.001). Both Croatian farmers and Bosnian immigrants observe significantly fewer Aristolochia plants growing in their fields compared with 15-30 years ago. Prior aristolochic acid exposure was associated with proximal tubule damage (odds ratio, 1.64; 95% confidence interval, 1.04 to 2.58; P=0.02), whereas present exposure was not (odds ratio, 1.31; 95% confidence interval, 0.75 to 2.30; P=0.33). Furthermore, immigrant status was an independent negative predictor of proximal tubule damage (odds ratio, 0.40; 95% confidence interval, 0.19 to 0.86; P=0.02). Bosnian immigrants and autochthonous Croats residing in endemic areas are exposed significantly less to ingestion of aristolochic acid than in the past. The prevalence of endemic nephropathy and its associated urothelial cancers is predicted to decrease over time.
Nephrology Dialysis Transplantation, 2013
Urologic Oncology: Seminars and Original Investigations, 2014
Upper tract urothelial carcinoma (UTUC) is a rare disease in Western countries and garners little... more Upper tract urothelial carcinoma (UTUC) is a rare disease in Western countries and garners little focused attention in urologic and oncologic circles. We report highlights from the first symposium on UTUC. All participants were asked to provide a summary of their presentation to be included as part of these proceedings. Submitted summaries were synthesized into this document. All contributors reviewed and provided input on the final draft. Five highlights are included in this report, including landmark research that not only reveals the likely cause of Balkan endemic nephropathy and associated UTUC but also links it directly to UTUC in Taiwan. Because of the ubiquitous use of Aristolochia plants in these herbal remedies, a public health problem of considerable magnitude is anticipated in Asian countries. Gene expression signatures reveal some differential expression in bladder carcinoma, such as CLCA2 and GABRE. Few urinary markers have proven utility for the diagnosis and follow-up of UTUC, and no tissue or blood-based markers are currently undergoing clinical application. Novel endoscopic therapies provide some hope of improving tissue sampling, diagnosis, and kidney-sparing therapeutics, but the greatest potential lies in improving clinical (preoperative) risk stratification, which is critically limited in this disease. Biomarkers, currently untested, hold promise in identifying patients most likely to benefit from perioperative chemotherapy and at high risk from cisplatin-induced nephrotoxicity. Despite its rarity in the West, UTUC is reaching potentially epidemic proportions in the East because of exposure to carcinogenic herbal remedies. Critical trials are needed to improve our understanding and treatment of UTUC. Because of the broad range of comorbid conditions in patients suffering from this disease, it is the consensus of the participants that future clinical trials should be practical in design and applicable to a broad range of patients, diverging from the current dogma of narrow patient selection criteria in clinical trials. Practical designs would maximize accrual for a still uncommon disease, and their findings would be applicable to a larger proportion of patients than current narrowly selected designs.
Mutation Research/Reviews in Mutation Research, 2013
Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental car... more Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental carcinogens. Here we review the pieces of evidence that support the role of aristolochic acid (AA) in inducing a mutational fingerprint in the tumor suppressor gene TP53 in urothelial carcinomas of the upper urinary tract (UUT). Exposure to AA, a nitrophenathrene carboxylic acid present in certain herbal remedies and in flour prepared from wheat grain contaminated with seeds of Aristolochia clematitis, has been linked to chronic nephropathy and UUT. TP53 mutations in UUT of individuals exposed to AA reveal a unique pattern of mutations characterized by A to T transversions on the non-transcribed strand, which cluster at hotspots rarely mutated in other cancers. This unusual pattern, originally discovered in UUTs from two different populations, one in Taiwan, and one in the Balkans, has been reproduced experimentally by treating mouse cells that harbor human TP53 sequences with AA. The convergence of molecular epidemiological and experimental data establishes a clear causal association between exposure to the human carcinogen AA and UUT. Despite bans on the sale of herbs containing AA, their use continues, raising global public health concern and an urgent need to identify populations at risk.
Journal of Molecular Modeling, 1999
DNA is a long, thread-like macromolecule composed of two helical polynucleotide chains. The chain... more DNA is a long, thread-like macromolecule composed of two helical polynucleotide chains. The chains are coiled around a common axis and are antisymmetric with respect to the helical axis. The most frequent conformation is a so-called B-DNA in which the diameter of the double helix is approximately 20 Å.