Biomedical subjects

Bongsup P Cho

Publications and source records attributed to Bongsup P Cho.

12 recordsLinked to original sources

PubMed2024

Comparative Studies on Bulky DNA Damage Binding by Nucleotide Excision Repair Proteins Using Surface Plasmon Resonance, Differential Scanning Fluorometry, and DNase I Footprinting.

Nucleotide excision repair is a crucial cellular mechanism that ensures genomic stability, thereby preventing mutations that can lead to cancer. The human XPC and its yeast ortholog Rad4 protein complexes are central to this process and were the focus of the study. We used surface plasmon resonance and differential scanning fluorimetry to study the binding characteristics of XPC and Rad4 when bound to the bulky cluster di-FAAF-containing 55-mer duplex DNA. Our findings revealed that XPC binds 10 times more significant affinity to control and di-FAAF-modified DNA than Rad4 with greater protein-DNA interactions. Differential scanning fluorimetry indicates that Rad4 causes comparatively more significant conformational changes upon complexation with the damaged DNA. We conducted DNase I footprinting of the Rad4/DNA complex for the first time by determining the regions protected from DNase I digestion. The DNA at the lesion is entirely resistant to digestion by DNase I in the absence of Rad4 several nucleotides to the 3'-side of the first FAAF lesion. The lack of DNase I cleavage at the lesions did not change upon adding Rad4. However, in the presence of Rad4, a footprint is observed on the 7-nucleotide region (5'-TGGTGAT-3') of the complementary strand to the 3' side of the lesion.

Surface Plasmon Resonance↗

PubMed2007

Conformation-specific recognition of carcinogen-DNA adduct in escherichia coli nucleotide excision repair.

We report a systematic and quantitative structure-function relationship study of the major N-[deoxyguanosin-8-yl]-2-aminofluorene adduct (AF) derived from the prototype carcinogen 2-aminofluorene and its derivatives. The AF adduct is known to exist in two distinct conformational motifs, depending upon the location of the hydrophobic fluorine moiety: major groove binding "B type" (B) conformation (AF-dGanti) and base-displaced "stacked" (S) conformation (AF-dGsyn). The AF-induced S/B conformational heterogeneity is sequence-dependent and follows a typical two-site dynamic chemical exchange. The population of S conformation decreases in the order of 3'-G > A > C > T, indicating the importance of the purine flanking bases in promoting the stacking structure. Line-shape analysis showed that the S/B interconversion energy barriers (DeltaG) are in the narrow 14-16 kcal/mol range. The energy differences of the two conformers are relatively small (<0.5 kcal/mol), suggesting a possibility for a facile adduct conformation switch in the active site of a polymerase. The S/B equilibrium modulates the efficiency of Escherichia coli UvrABC-based nucleotide excision repair (NER) in a conformation-specific manner. The 19F NMR/NER results indicate greater repair susceptibility for the base-displaced S conformer, which lacks a Watson-Crick base pair at the lesion site. These findings represent the first of its kind quantitative structure-function work relating NER activity to a specific adduct conformer and will lead to a better understanding of how bulky DNA adducts are accommodated by the repair protein.

Base Sequence↗

PubMed2006

Examination of the long-range effects of aminofluorene-induced conformational heterogeneity and its relevance to the mechanism of translesional DNA synthesis.

Adduct-induced conformational heterogeneity complicates the understanding of how DNA adducts exert mutation. A case in point is the N-deacetylated AF lesion [N-(2'-deoxyguanosin-8-yl)-2-aminofluorene], the major adduct derived from the strong liver carcinogen N-acetyl-2-aminofluorene. Three conformational families have been previously characterized and are dependent on the positioning of the aminofluorene rings: B is in the "B-DNA" major groove, S is "stacked" into the helix with base-displacement, and W is "wedged" into the minor groove. Here, we conducted (19)F NMR, CD, T(m), and modeling experiments at various primer positions with respect to a template modified by a fluorine tagged AF-adduct (FAF). In the first set, the FAF-G was paired with C and in the second set it was paired with A. The FAF-G:C oligonucleotides were found to preferentially adopt the B or S-conformers while the FAF-G:A mismatch ones preferred the B and W-conformers. The conformational preferences of both series were dependent on temperature and complementary strand length; the largest differences in conformation were displayed at lower temperatures. The CD and T(m) results are in general agreement with the NMR data. Molecular modeling indicated that the aminofluorene moiety in the minor groove of the W-conformer would impose a steric clash with the tight-packing amino acid residues on the DNA binding area of the Bacillus fragment (BF), a replicative DNA polymerase. In the case of the B-type conformer, the carcinogenic moiety resides in the solvent-exposed major groove throughout the replication/translocation process. The present dynamic NMR results, combined with previous primer extension kinetic data by Miller & Grollman, support a model in which adduct-induced conformational heterogeneities at positions remote from the replication fork affect polymerase function through a long-range DNA-protein interaction.

Base Pair Mismatch↗

PubMed2006

Sequence effects of aminofluorene-modified DNA duplexes: thermodynamic and circular dichroism properties.

Circular dichroism (CD) and UV-melting experiments were conducted with 16 oligodeoxynucleotides modified by the carcinogen 2-aminofluorene, whose sequence around the lesion was varied systematically [d(CTTCTNG[AF]NCCTC), N = G, A, C, T], to gain insight into the factors that determine the equilibrium between base-displaced stacked (S) and external B-type (B) duplex conformers. Differing stabilities among the duplexes can be attributed to different populations of S and B conformers. The AF modification always resulted in sequence-dependent thermal (T(m)) and thermodynamic (-DeltaG degrees ) destabilization. The population of B-type conformers derived from eight selected duplexes (i.e. -AG*N- and -CG*N-) was inversely proportional to the -DeltaG degrees and T(m) values, which highlights the importance of carcinogen/base stacking in duplex stabilization even in the face of disrupted Watson-Crick base pairing in S-conformation. CD studies showed that the extent of the adduct-induced negative ellipticities in the 290-350 nm range is correlated linearly with -DeltaG degrees and T(m), but inversely with the population of B-type conformations. Taken together, these results revealed a unique interplay between the extent of carcinogenic interaction with neighboring base pairs and the thermodynamic properties of the AF-modified duplexes. The sequence-dependent S/B heterogeneities have important implications in understanding how arylamine-DNA adducts are recognized in nucleotide excision repair.

Base Sequence↗

PubMed2006

Synthesis and characterization of enantiomeric anti-2-fluorobenzo[a]pyrene-7,8-dihydrodiol-9,10-epoxides and their 2'-deoxyguanosine and oligodeoxynucleotide adducts.

Benzo[a]pyrene diol epoxides (BPDEs) are the ultimate carcinogenic species of benzo[a]pyrene, a prototype polycyclic aromatic hydrocarbon (PAH). BPDE-modified DNA duplexes can adopt multiple conformations depending on the nature of the modified bases, the stereochemistry at the location of the covalent linkage, and the sequence context surrounding the lesion site. In this paper, we describe the preparation of enantiomeric 2-fluoro-BPDEs, trans-(7R,8S)-dihydroxy-(9S,10R)- and trans-(7S,8R)-(9R,10S)-epoxy-7,8,9,10-tetrtahydro-2-fluorobenzo[a]pyrene (22 and 23, respectively), as models for probing the BPDE-induced conformational heterogeneity. The multistep synthesis of the target diol epoxides described herein entails regiospecific succinoylation of 2-fluoropyrene, followed by a ring closure, regio- and stereospecific construction of the 7,8-dihydrodiol functionality, and a subsequent meta-chloroperbenzoic acid-mediated epoxidation. Stereoselectivity was achieved by using Jacobsen chiral catalysts, which produced greater than approximately 90% enantiomeric excess. Absolute configurations at the C(7,8) carbons of the FBP derivatives were determined by comparison of the circular dichroism (CD) spectra with those reported for the BP analogues. Analysis of the 3J(7,8) vicinal coupling constants, CD shape, and charge density calculations all indicated that the prepared anti-FBPDEs preferentially adopt the pseudo-diequatorial C(7,8) diol conformation. Hydrolysis of anti-FBPDEs produced a 9:1 ratio of trans- to cis-opened tetraols. Reactions of each of the anti-FBPDEs with deoxyguanosine 5'-monophosphate produced predominantly trans-anti-N2-dG as the major adducts. Analogous reactions with two 11-mer oligodeoxynucleotides (5'-CCATXGCTACC-3' where X = dT, dC) gave FBP-modified oligodeoxynucleotides with structures that were characterized by enzyme digest/HPLC and electrospray ionization time-of-flight mass spectrometry data. The oligonucleotide adducts were annealed with the appropriate sequences to form fully complementary duplexes [(5'-CCATXGCTACC-3')(5'-GGTAGCYATGG-3'), G = FBP-N2-dG adduct, X = dT, Y = dA in duplex I; X = dC, Y = dG in duplex II] for CD and UV melting studies. The results of the present study were consistent with those reported previously for BPDE-modified duplexes in the same sequence contexts and support the utility of FBPDEs as useful structural probes.

DNA Adducts↗

PubMed2006

Induced circular dichroism characteristics as conformational probes for carcinogenic aminofluorene-DNA adducts.

We report novel induced circular dichroism (ICD) characteristics for probing the conformational heterogeneity induced by the arylamine carcinogen 2-aminofluorene, namely, B type (B), stacked (S), and wedged (W) conformers. CD experiments were conducted with five different aminofluorene-modified DNA duplexes (I-V). An intense positive ICD was observed for the W conformeric I in the 290-360 nm range (ICD(290)(-)(360nm)). This was in contrast to the negative ICD(290)(-)(360nm) exhibited by the mostly B conformeric V (17% S/83% B). Duplex IV, which adopts an approximately equal mixture of S (53%) and B (47%), exhibited low ellipticities along the baseline. The magnitude of the positive ICD for I was significantly greater than that observed for II (70% S/30% B). While the ICD(290)(-)(360nm) of the W conformeric III showed no changes in intensity with increasing temperature from 10 to 35 degrees C, dramatic changes were observed for I across the same temperature range. Dynamic (19)F NMR results revealed that I exists in an 85:15 mixture of W and S/B conformers. The dramatic intensity changes observed for I are consistent with the presence of a W/B heterogeneity because of its susceptibility to result in a large difference on the magnitude of the ICD(290)(-)(360nm). In conclusion, the sign and magnitude of the ICD(290)(-)(360)(nm) are sensitive conformational markers for studying arylamine-induced conformational heterogeneity. The temperature-dependent ICD(290)(-)(360nm) data, coupled with (19)F NMR spectroscopy, provide valuable information about conformational distribution and dynamics, which are important factors that affect mutational outcomes.

Binding Sites↗

PubMed2005

1H NMR analysis of GHB and GBL: further findings on the interconversion and a preliminary report on the analysis of GHB in serum and urine.

A 1H nuclear magnetic resonance (1H NMR) method for the determination of gamma-hydroxybutyric acid (GHB) and gamma-hydroxybutyrolactone (GBL) in human serum and urine using spiked samples has been developed. The method gives linear responses (correlation coefficients of 0.99 or greater) over the concentration range 0.01 mg/mL to 4.0 mg/mL in urine and 0.3 mg/mL to 2.0 mg/mL in serum. No sample pretreatment is required. Studies of the chemical interconversion of GBL and GHB showed hydrolysis of GBL to be rapid at pH 11.54, slower and less complete (30% hydrolysis) at pH 2.54 and slowest at pH 7.0, reaching 30% hydrolysis in about 40 days. No esterification of GHB was observed at any pH.

4-Butyrolactone↗

PubMed2004

Dynamic conformational heterogeneities of carcinogen-DNA adducts and their mutagenic relevance.

Arylamines and polycyclic aromatic hydrocarbons (PAHs), which are known as "bulky" carcinogens, have been studied extensively and upon activation in vivo, react with cellular DNA to form DNA-adducts. The available structure data accumulated thus far has revealed that conformational heterogeneity is a common theme among duplex DNA modified with these carcinogens. Several conformationally diverse structures have been elucidated and found to be in equilibrium in certain cases. The dynamics of the heterogeneity appear to be modulated by the nature of the adduct structure and the base sequences neighboring the lesion site. These can be termed as "adduct- and sequence-induced conformational heterogeneities," respectively. Due to the small energy differences, the population levels of these conformers could readily be altered within the active sites of repair or replicate enzymes. Thus, the complex role of "enzyme-induced conformational heterogeneity" must also be taken into consideration for the establishment of a functional structure-mutation relationship. Ultimately, a major challenge in mutation structural biology is to carry out adduct- and site-specific experiments in a conformationally specific manner within biologically relevant environments. Results from such experiments should provide an accurate account of how a single chemically homogenous adduct gives rise to complex multiple mutations, the earliest step in the induction of cancer.

Carcinogens↗

PubMed2003

Synthesis and characterization of N-demethylated metabolites of malachite green and leucomalachite green.

Malachite green (MG), a triphenylmethane dye used to treat fungal and protozoan infections in fish, undergoes sequential oxidation to produce various N-demethylated derivatives (monodes-, dides(sym)-, dides(unsym)-, trides-, and tetrades-) both before and after reduction to leucomalachite green (LMG). The close structure resemblance of the metabolites with aromatic amine carcinogens implicates a potential genotoxicity from exposure to MG. The availability of the synthetic standards is important for metabolic and DNA adduct studies of MG. This paper describes a simple and versatile method for the synthesis of MG, LMG, and their N-demethylated metabolites. The synthesis involves a coupling of 4-(dimethylamino)benzophenone or 4-nitrobenzophenone with the aryllithium reagents derived from appropriately substituted 4-bromoaniline derivatives, followed by treatment with HCl in methanol. The resulting cationic MG and their leuco analogues showed systematic UV/vis spectral and tandem mass fragmentation patterns consistent with sequential N-demethylation. The extensive (1)H and (13)C spectral assignments of the metabolites were aided by the availability of (13)C(7)-labeled MG and LMG. The results indicate the existence of a resonance structure with the cationic charge located in the central methane carbon (C(7)). The synthetic procedure is general in scope so that it can be extended to the preparation of N-demethylated metabolites of other structurally related N-methylated triphenylmethane dyes.

Aniline Compounds↗

PubMed2003

Structural differentiation of diastereomeric benzo[ghi]fluoranthene adducts of deoxyadenosine by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and postsource decay.

The product ion formation characteristics of four diastereomeric deoxyadenosine adducts formed by the reaction of the syn and anti diastereomers of trans-3,4-dihydroxy-5,5a-epoxy-3,4,5,5a-tetrahydrobenzo[ghi]fluoranthene are studied by matrix-assisted laser desorption ionization and postsource decay (PSD) to determine fragmentation pathways that may permit differentiation of their structures. The two adducts derived from each diol-epoxide with DNA differ in structure based on the cis/trans arrangement of the 3'-hydroxyl group on the benzo[ghi]fluoranthene (B[ghi]F) and the adenine base bound to the B[ghi]F 5a carbon. The two adduct diastereomers with the cis adenine-3'-hydroxyl configuration produce product ions at m/z 394 and m/z 510 formed by the loss of water that are not observed in the PSD spectra of the two trans isomers. The data suggest a mechanism of water loss that is initiated by a hydrogen-bonding interaction between the charge-bearing proton on the N1 atom and the 3'-hydroxyl oxygen on the polycyclic aromatic hydrocarbon (PAH). Fragmentation is initiated by the transfer of the adenine N1 proton from the nitrogen to the PAH 3'-hydroxyl oxygen and inductive cleavage of the C3-O(3) bond to form a benzylic carbocation on B[ghi]F. The proposed mechanism is supported by semiempirical molecular modeling calculations.

Adenine↗

PubMed2002

Characterization of DNA adducts and tetraols derived from anti-benzo[ghi]fluoranthane-3,4-dihydrodiol-5,5a-epoxide.

A total of seven DNA adducts and two racemic tetraols derived from anti-benzo[ghi]fluoranthene-3,4-dihydrodiol-5,5a-epoxide (anti-B[ghi]FDE, 2) were characterized by analyses of UV, (1)H NMR, CD, and MALDI mass spectra. The structure of 2 is the first example of a diolepoxide in which a fully fused cyclopentane ring is covalently linked to the saturated ring bearing the epoxide function. Compound 2 is also a conformationally rigid structure analogue of the extensively studied anti-benzo[c]phenanthrene-3,4-dihydrodiol-1,2-epoxide (anti-BcPDE), thus serving as a model for probing the diolepoxide-DNA interaction [Chang et al. (2002) Chem. Res. Toxicol. 15, 198-208 (following paper in this issue)]. The most abundant adducts are formed from trans- or cis-openings of the epoxide by the amino groups of either deoxyguanosine or deoxyadenosine. Adducts of minor abundance formed by the attachment of the diolepoxide to the amino group of deoxycytidine N(4) and guanine N(7) were also isolated. Post-source decay MALDI spectra of the (M + H)(+) molecule ions are consistent with the assigned adduct structures. The lack of a typical benzylic proton at the site of deoxynucleoside attachment necessitated a new NMR assignment strategy. Despite the steric constraint, the epoxide ring opening of 2 occurred exclusively at the dibenzylic C5a, not at C5. The assignments on the trans- and cis-epoxide opening were made based on the molecular modeling structures, i.e., the pseudoaxial H5 in cis-adducts is placed directly under the strong influence of a shielding cone of the aromatic ring system, while the same proton in trans-adducts adopts a pseudoequatorial conformation, thereby protruding away from the aromatic ring system. The absolute configuration at the site of deoxynucleoside attachment (C5a) was tentatively assigned on the basis of the empirical rules that have been established for deoxynucleoside-adducts derived from traditional alternant PAH diolepoxides.

Animals↗

PubMed2002

Characterization of DNA adducts derived from syn-benzo[ghi]fluoranthene-3,4-dihydrodiol-5,5a-epoxide and comparative DNA binding studies with structurally-related anti-diolepoxides of benzo[ghi]fluoranthene and benzo[c]phenanthrene.

This paper reports structural characterization of the adducts and tetraols formed from syn-benzo[ghi]fluoranthene-3,4-dihydrodiol-5,5a-epoxide (syn-B[ghi]FDE, 3) and comparative DNA-binding and mutagenicity studies involving 3, anti-B[ghi]FDE (2), and anti-benzo[c]phenanthrene-11,12-dihydrodiol-13,14-epoxide (anti-BcPDE, 5). The structures of nine DNA adducts and two racemic tetraols derived from 3 have been determined spectroscopically. Similar characterization of adducts obtained from the anti-isomer 2 was described in the preceding paper in this issue [Chang et al. (2002) Chem. Res. Toxicol. 15, 187-197]. The majority of DNA adducts with 3 are those from the trans- or cis-opening of the epoxide at C5a by the exocyclic amino groups of dG, dA, and dC. The diolepoxides 2 and 3 are rigid structure analogues of anti- and syn-BcPDE (5 and 6), respectively, thus serving as models for probing molecular deformity and diol conformation in diolepoxide-DNA interaction. Comparative DNA binding experiments indicate that 57% of 2 and 33% of 3 were converted into DNA adducts, whereas a 71% conversion was observed for 5. In general, lower percentages were observed with denatured calf-thymus DNA. As for base selectivity, 2 showed a greater affinity for dA relative to dG (dA/dG ratio, 0.79) than 3 (0.56) when reacted with native calf-thymus DNA. A much higher dA/dG ratio (1.41) was obtained for 5. The overall dA/dG ratios were lower with denatured DNA, indicating the importance of the secondary structure of DNA for both adduct formation and chemical selectivity. The T-shape pseudo-diaxial diols of 3 appears to have favorable electrostatic interactions with the nearby phosphate backbone in the minor groove of DNA, thereby yielding greater amounts of dG adducts than the pseudo-diequatorial 2. The anti-isomer 2 was found to be seven times more mutagenic than 3, but they are significantly less mutagenic than the nonplanar analogue 5 when tested in Salmonella typhimurium TA 100.

Animals↗