Susith Wickramaratne - Academia.edu (original) (raw)

Papers by Susith Wickramaratne

Research paper thumbnail of Structure of Exocyclic R,R N6,N6-(2,3-Dihydroxy-1,4-butadiyl)-2'-Deoxyadenosine Adduct Induced by 1,2,3,4-Diepoxybutane in DNA

Research paper thumbnail of Synthesis of Site-Specific DNA−Protein Conjugates and Their Effects on DNA Replication

ABSTRACT: DNA−protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the... more ABSTRACT: DNA−protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupa-tional toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA−protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subj...

Research paper thumbnail of Synthesis and biological evaluation of pyrrolidine-functionalized nucleoside analogs

Medicinal Chemistry Research, 2021

Inhibition of viral reverse transcriptases and mammalian DNA polymerases by unnatural nucleoside ... more Inhibition of viral reverse transcriptases and mammalian DNA polymerases by unnatural nucleoside analogs is a proven approach in antiviral and anticancer therapy, respectively. The majority of current nucleoside drugs retain the canonical nucleobase structure, which is fused to an unnatural sugar. In the present work, a series of novel pyrrolidine-functionalized purine and pyrimidine nucleosides was prepared via PyBOP-catalyzed SNAr addition-elimination reactions of commercial halogenated precursors and tested for their antiviral and anticancer activity. The newly synthesized nucleoside analogs showed limited biological activity, probably as a result of their poor cellular uptake and their inefficient bioactivation to the corresponding nucleoside monophosphates. A phosphoramidate prodrug had an improved cell permeability and was metabolized to the nucleoside monophosphate form in human cells, as revealed by HPLC-MS/MS analyses.

Research paper thumbnail of pH dependence of bentonite aggregate size and morphology on polymer-clay flocculation

Colloids and Surfaces A: Physicochemical and Engineering Aspects

Research paper thumbnail of Ionic strength dependence of aggregate size and morphology on polymer-clay flocculation

Colloids and Surfaces A: Physicochemical and Engineering Aspects

Research paper thumbnail of Cellular Repair of DNA-DNA Cross-Links Induced by 1,2,3,4-Diepoxybutane

International journal of molecular sciences, Jan 18, 2017

Xenobiotic-induced interstrand DNA-DNA cross-links (ICL) interfere with transcription and replica... more Xenobiotic-induced interstrand DNA-DNA cross-links (ICL) interfere with transcription and replication and can be converted to toxic DNA double strand breaks. In this work, we investigated cellular responses to 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links induced by 1,2,3,4-diepoxybutane (DEB). High pressure liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI⁺-MS/MS) assays were used to quantify the formation and repair of bis-N7G-BD cross-links in wild-type Chinese hamster lung fibroblasts (V79) and the corresponding isogenic clones V-H1 and V-H4, deficient in the XPD and FANCA genes, respectively. Both V-H1 and V-H4 cells exhibited enhanced sensitivity to DEB-induced cell death and elevated bis-N7G-BD cross-links. However, relatively modest increases of bis-N7G-BD adduct levels in V-H4 clones did not correlate with their hypersensitivity to DEB. Further, bis-N7G-BD levels were not elevated in DEB-treated human clones with defects in the X...

Research paper thumbnail of Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

The Journal of biological chemistry, Jan 12, 2016

DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following ex... more DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis (TLS) DNA polymerases. DPCs containing full length proteins (11-28 kDa) or a 23-me...

Research paper thumbnail of Internal structure visualization of polymer — clay flocculants using fluorescence

Colloid and Interface Science Communications, 2016

Research paper thumbnail of Base Excision Repair of N(6)-Deoxyadenosine Adducts of 1,3-Butadiene

Biochemistry, Jan 23, 2016

The important industrial and environmental carcinogen, 1,3-butadiene (BD), forms a range of adeni... more The important industrial and environmental carcinogen, 1,3-butadiene (BD), forms a range of adenine adducts in DNA, including N(6)-(2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N(6)-HB-dA), 1,N(6)-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-HMHP-dA), and N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N(6),N(6)-DHB-dA). If not removed prior to DNA replication, these lesions can contribute to A → T and A → G mutations commonly observed following exposure to BD and its metabolites. In the present study, base excision repair of BD-induced 2'-deoxyadenosine (BD-dA) lesions was investigated. Synthetic DNA duplexes containing site- and stereospecific S-N(6)-HB-dA, R,S-1,N(6)-HMHP-dA, and R,R-N(6),N(6)-DHB-dA adducts were prepared by a post-oligomerization strategy. Incision assays with nuclear extracts from human fibrosarcoma (HT1080) cells have revealed that BD-dA adducts were recognized and cleaved by a BER mechanism, with relative excis...

Research paper thumbnail of Polymerase Bypass of N 6 -Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene

Chemical Research in Toxicology, 2015

N(6)-(2-Hydroxy-3-buten-1-yl)-2&a... more N(6)-(2-Hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N(6)-HB-dA I) and N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N(6),N(6)-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the N(6) position of adenine in DNA by epoxide metabolites of 1,3-butadiene (BD), a common industrial and environmental chemical classified as a human and animal carcinogen. Since the N(6)-H atom of adenine is required for Watson-Crick hydrogen bonding with thymine, N(6)-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived N(6)-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (S)-N(6)-HB-dA I and (R,R)-N(6),N(6)-DHB-dA adducts were subjected to in vitro translesion synthesis in the presence of human DNA polymerases β, η, ι, and κ. While (S)-N(6)-HB-dA I was readily bypassed by all four enzymes, only polymerases η and κ were able to carry out DNA synthesis past (R,R)-N(6),N(6)-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (S)-N(6)-HB-dA I. In contrast, hPol β was completely blocked by (R,R)-N(6),N(6)-DHB-dA, while hPol η and κ inserted A, G, C, or T opposite the adduct with similar frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed that while translesion synthesis past (S)-N(6)-HB-dA I was mostly error-free, replication of DNA containing (R,R)-N(6),N(6)-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (S)-N(6)-HB-dA I lesions are not miscoding, but that exocyclic (R,R)-N(6),N(6)-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson-Crick pairs with dT.

Research paper thumbnail of Major Groove Orientation of the (2 S )- N 6 -(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine DNA Adduct Induced by 1,2-Epoxy-3-butene

Chemical Research in Toxicology, 2014

1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen... more 1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. It is oxidized by cytochrome P450 monooxygenases to 1,2-epoxy-3butene (EB), which alkylates DNA. BD exposures lead to large numbers of mutations at A:T base pairs even though alkylation of guanines is more prevalent, suggesting that one or more adenine adducts of BD play a role in BD-mediated genotoxicity. However, the etiology of BD-mediated genotoxicity at adenine remains poorly understood. EB alkylates the N 6 exocyclic nitrogen of adenine to form N 6-(hydroxy-3buten-1-yl)-2′-dA ((2S)-N 6-HB-dA) adducts (

Research paper thumbnail of NanoLC/ESI+ HRMS3 Quantitation of DNA Adducts Induced by 1,3-Butadiene

Journal of The American Society for Mass Spectrometry, 2014

Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest f... more Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest fires is of great concern because of its potent carcinogenicity. The adverse health effects of BD are mediated by its epoxide metabolites such as 3,4-epoxy-1-butene (EB), which covalently modify genomic DNA to form promutagenic nucleobase adducts. Because of their direct role in cancer, BD-DNA adducts can be used as mechanism-based biomarkers of BD exposure. In the present work, a mass spectrometry-based methodology was developed for accurate, sensitive, and precise quantification of EB-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) DNA adducts in vivo. In our approach, EB-GII adducts are selectively released from DNA backbone by neutral thermal hydrolysis, followed by ultrafiltration, offline HPLC purification, and isotope dilution nanoLC/ESI(+)-HRMS(3) analysis on an Orbitrap Velos mass spectrometer. Following method validation, EB-GII lesions were quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of EB and in liver tissues of rats exposed to sub-ppm concentrations of BD (0.5-1.5 ppm). EB-GII concentrations increased linearly from 1.15 ± 0.23 to 10.11 ± 0.45 adducts per 10(8) nucleotides in HT1080 cells treated with 0.5-10 μM EB. EB-GII concentrations in DNA of laboratory rats exposed to 0.5, 1.0, and 1.5 ppm BD were 0.17 ± 0.05, 0.33 ± 0.08, and 0.50 ± 0.04 adducts per 10(8) nucleotides, respectively [corrected]. We also used the new method to determine the in vivo half-life of EB-GII adducts in rat liver DNA (2.20 ± 0.12 d) and to detect EB-GII in human blood DNA. To our knowledge, this is the first application of nanoLC/ESI(+)-HRMS(3) Orbitrap methodology to quantitative analysis of DNA adducts in vivo.

Research paper thumbnail of Erratum to: NanoLC/ESI+ HRMS3 Quantitation of DNA Adducts Induced by 1,3-Butadiene

Journal of The American Society for Mass Spectrometry, 2014

in our recently published article contains several errors that were introduced during article pro... more in our recently published article contains several errors that were introduced during article production. The adduct numbers should be expressed "per 10 8 nucleotides" rather than "per 10 6 nucleotides." Also, "0.5-10 μM DEB" should be read "0.5-10 μM EB.

Research paper thumbnail of Synthesis of Site-Specific DNA–Protein Conjugates and Their Effects on DNA Replication

ACS Chemical Biology, 2014

DNA−protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome up... more DNA−protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radicalgenerating systems. As a result of their considerable size and their pronounced effects on DNA−protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts.

Research paper thumbnail of Translesion Synthesis across 1,N6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6- -HMHP-dA) Adducts by Human and Archebacterial DNA Polymerases

Journal of Biological Chemistry, 2012

Background: 1,N 6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2Ј-deoxyadenosine (1,N 6-␥-HMHP-dA) ... more Background: 1,N 6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2Ј-deoxyadenosine (1,N 6-␥-HMHP-dA) adducts are formed in DNA by 1,2,3,4-diepoxybutane (metabolite of human carcinogen 1,3-butadiene). Results: hpols and carry out translesion synthesis, incorporating T, G, or A opposite the 1,N 6-␥-HMHP-dA adduct. Conclusion: Translesion bypass of 1,N 6-␥-HMHP-dA adducts by TLS polymerases is error-prone. Significance: This study identifies 1,N 6-␥-HMHP-dA as the DNA adduct potentially responsible for A3 T and A3 C transversions and deletions induced by 1,3-butadiene. The 1,N 6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2deoxyadenosine (1,N 6-␥-HMHP-dA) adducts are formed upon bifunctional alkylation of adenine nucleobases in DNA by 1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene. The presence of a substituted 1,N 6propano group on 1,N 6-␥-HMHP-dA is expected to block the Watson-Crick base pairing of the adducted adenine with thymine, potentially contributing to mutagenesis. In this study, the enzymology of replication past site-specific 1,N 6-␥-HMHP-dA lesions in the presence of human DNA polymerases (hpols) ␤, , , and and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol , , and Dpo4 were able to copy the modified template. In contrast, hpol inserted a single base opposite 1,N 6-␥-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol ␤. Single nucleotide incorporation experiments indicated that although hpol , , and Dpo4 incorporated the correct nucleotide (dTMP) opposite the lesion, dGMP and dAMP were inserted with a comparable frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed the ability of bypass polymerases to insert dTMP, dAMP, or dGMP opposite 1,N 6-␥-HMHP-dA and detected large amounts of ؊1 and ؊2 deletion products. Taken together, these results indicate that hpol and enzymes bypass 1,N 6-␥-HMHP-dA lesions in an error-prone fashion, potentially contributing to A3 T and A3 C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane.

Research paper thumbnail of Synthesis of DNA oligodeoxynucleotides containing structurally defined N6-(2-hydroxy-3-buten-1-yl)-adenine adducts of 3,4-epoxy-1-butene

Chemico-Biological Interactions, 2007

3,4-Epoxy-1-butene (EB) is generated by cytochrome P450-mediated epoxidation of 1,3-butadiene (BD... more 3,4-Epoxy-1-butene (EB) is generated by cytochrome P450-mediated epoxidation of 1,3-butadiene (BD), an important environmental and industrial chemical classified as a probable human carcinogen. The ability of EB to induce point mutations at GC and AT base pairs has been attributed to its reactions with DNA to form covalent nucleobase adducts. Guanine alkylation is preferred at the endocyclic N7 nitrogen, while adenine can be modified at the N1-, N3-, N7-, and the N 6 positions. For each of these sites, a pair of regioisomeric 2-hydroxy-3-buten-1-yl and 1-hydroxy-3-buten-2-yl adducts is produced as a result of epoxide ring opening at the terminal C-4 or the internal C-3 carbon position of EB, respectively. The N 6-EB-adenine adducts are of particular interest because of their stability in DNA, potentially leading to their accumulation in vivo. In the present work, synthetic DNA oligomers containing structurally defined N 6-(2-hydroxy-3-buten-1-yl)-dA (N 6-HB-dA) adducts were prepared for the first time by a postoligomerization approach that involved coupling 6-chloropurine-containing DNA with synthetic 1-amino-3-buten-2-ol. N 6-HB-dA-containing DNA oligomers were isolated by reversed phase HPLC, and the presence of N 6-HB-dA in their structure was confirmed by molecular weight determination from HPLC-ESI −-MS of the intact strands and by HPLC-ESI +-MS/MS and MS/MS/MS analyses of the enzymatic digests using synthetic N 6-HB-dA as an authentic standard. N 6-HB-dA-containing oligomers generated in this study will be used for structural and biological studies.

Research paper thumbnail of Structures of Exocyclic R,R - and S,S - N 6 , N 6 -(2,3-Dihydroxybutan-1,4-diyl)-2′-Deoxyadenosine Adducts Induced by 1,2,3,4-Diepoxybutane

Chemical Research in Toxicology, 2014

1,3-Butadiene (BD) is an industrial and environmental chemical present in urban air and cigarette... more 1,3-Butadiene (BD) is an industrial and environmental chemical present in urban air and cigarette smoke, and is classified as a human carcinogen. It is oxidized by cytochrome P450 to form 1,2,3,4-diepoxybutane (DEB); DEB bis-alkylates the N 6 position of adenine in DNA. Two enantiomers of bis-N 6-dA adducts of DEB have been

Research paper thumbnail of Structure Elucidation of DNA-Protein Crosslinks by Using Reductive Desulfurization and Liquid Chromatography-Tandem Mass Spectrometry

ChemBioChem, 2014

Easier with ethyl: Guengerich and co-workers have developed a powerful new approach to the struct... more Easier with ethyl: Guengerich and co-workers have developed a powerful new approach to the structure elucidation of hydrolytically stable AGT-DNA crosslinks by reductive desulfurization of the thioether linkage between AGT and DNA to convert cysteine DPCs to the corresponding ethyl-DNA adducts, which can be readily characterized by LC-MSn.

Research paper thumbnail of Synthesis of Sequence-Specific DNA–Protein Conjugates via a Reductive Amination Strategy

Bioconjugate Chemistry, 2013

DNA-protein cross-links (DPCs) are ubiquitous, structurally diverse DNA lesions formed upon expos... more DNA-protein cross-links (DPCs) are ubiquitous, structurally diverse DNA lesions formed upon exposure to bis-electrophiles, transition metals, UV light, and reactive oxygen species. Because of their superbulky, helix distorting nature, DPCs interfere with DNA replication, transcription, and repair, potentially contributing to mutagenesis and carcinogenesis. However, the biological implications of DPC lesions have not been fully elucidated due to the difficulty in generating site-specific DNA substrates representative of DPC lesions formed in vivo. In the present study, a novel approach involving postsynthetic reductive amination has been developed to prepare a range of hydrolytically stable lesions structurally mimicking the DPCs produced between the N7 position of guanine in DNA and basic lysine or arginine side chains of proteins and peptides.

Research paper thumbnail of DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells

Biochemistry, 2013

Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to ... more Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions, including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O(6)-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Smaller numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles.

Research paper thumbnail of Structure of Exocyclic R,R N6,N6-(2,3-Dihydroxy-1,4-butadiyl)-2'-Deoxyadenosine Adduct Induced by 1,2,3,4-Diepoxybutane in DNA

Research paper thumbnail of Synthesis of Site-Specific DNA−Protein Conjugates and Their Effects on DNA Replication

ABSTRACT: DNA−protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the... more ABSTRACT: DNA−protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupa-tional toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA−protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subj...

Research paper thumbnail of Synthesis and biological evaluation of pyrrolidine-functionalized nucleoside analogs

Medicinal Chemistry Research, 2021

Inhibition of viral reverse transcriptases and mammalian DNA polymerases by unnatural nucleoside ... more Inhibition of viral reverse transcriptases and mammalian DNA polymerases by unnatural nucleoside analogs is a proven approach in antiviral and anticancer therapy, respectively. The majority of current nucleoside drugs retain the canonical nucleobase structure, which is fused to an unnatural sugar. In the present work, a series of novel pyrrolidine-functionalized purine and pyrimidine nucleosides was prepared via PyBOP-catalyzed SNAr addition-elimination reactions of commercial halogenated precursors and tested for their antiviral and anticancer activity. The newly synthesized nucleoside analogs showed limited biological activity, probably as a result of their poor cellular uptake and their inefficient bioactivation to the corresponding nucleoside monophosphates. A phosphoramidate prodrug had an improved cell permeability and was metabolized to the nucleoside monophosphate form in human cells, as revealed by HPLC-MS/MS analyses.

Research paper thumbnail of pH dependence of bentonite aggregate size and morphology on polymer-clay flocculation

Colloids and Surfaces A: Physicochemical and Engineering Aspects

Research paper thumbnail of Ionic strength dependence of aggregate size and morphology on polymer-clay flocculation

Colloids and Surfaces A: Physicochemical and Engineering Aspects

Research paper thumbnail of Cellular Repair of DNA-DNA Cross-Links Induced by 1,2,3,4-Diepoxybutane

International journal of molecular sciences, Jan 18, 2017

Xenobiotic-induced interstrand DNA-DNA cross-links (ICL) interfere with transcription and replica... more Xenobiotic-induced interstrand DNA-DNA cross-links (ICL) interfere with transcription and replication and can be converted to toxic DNA double strand breaks. In this work, we investigated cellular responses to 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links induced by 1,2,3,4-diepoxybutane (DEB). High pressure liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI⁺-MS/MS) assays were used to quantify the formation and repair of bis-N7G-BD cross-links in wild-type Chinese hamster lung fibroblasts (V79) and the corresponding isogenic clones V-H1 and V-H4, deficient in the XPD and FANCA genes, respectively. Both V-H1 and V-H4 cells exhibited enhanced sensitivity to DEB-induced cell death and elevated bis-N7G-BD cross-links. However, relatively modest increases of bis-N7G-BD adduct levels in V-H4 clones did not correlate with their hypersensitivity to DEB. Further, bis-N7G-BD levels were not elevated in DEB-treated human clones with defects in the X...

Research paper thumbnail of Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

The Journal of biological chemistry, Jan 12, 2016

DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following ex... more DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis (TLS) DNA polymerases. DPCs containing full length proteins (11-28 kDa) or a 23-me...

Research paper thumbnail of Internal structure visualization of polymer — clay flocculants using fluorescence

Colloid and Interface Science Communications, 2016

Research paper thumbnail of Base Excision Repair of N(6)-Deoxyadenosine Adducts of 1,3-Butadiene

Biochemistry, Jan 23, 2016

The important industrial and environmental carcinogen, 1,3-butadiene (BD), forms a range of adeni... more The important industrial and environmental carcinogen, 1,3-butadiene (BD), forms a range of adenine adducts in DNA, including N(6)-(2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N(6)-HB-dA), 1,N(6)-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-HMHP-dA), and N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N(6),N(6)-DHB-dA). If not removed prior to DNA replication, these lesions can contribute to A → T and A → G mutations commonly observed following exposure to BD and its metabolites. In the present study, base excision repair of BD-induced 2'-deoxyadenosine (BD-dA) lesions was investigated. Synthetic DNA duplexes containing site- and stereospecific S-N(6)-HB-dA, R,S-1,N(6)-HMHP-dA, and R,R-N(6),N(6)-DHB-dA adducts were prepared by a post-oligomerization strategy. Incision assays with nuclear extracts from human fibrosarcoma (HT1080) cells have revealed that BD-dA adducts were recognized and cleaved by a BER mechanism, with relative excis...

Research paper thumbnail of Polymerase Bypass of N 6 -Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene

Chemical Research in Toxicology, 2015

N(6)-(2-Hydroxy-3-buten-1-yl)-2&a... more N(6)-(2-Hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N(6)-HB-dA I) and N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N(6),N(6)-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the N(6) position of adenine in DNA by epoxide metabolites of 1,3-butadiene (BD), a common industrial and environmental chemical classified as a human and animal carcinogen. Since the N(6)-H atom of adenine is required for Watson-Crick hydrogen bonding with thymine, N(6)-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived N(6)-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (S)-N(6)-HB-dA I and (R,R)-N(6),N(6)-DHB-dA adducts were subjected to in vitro translesion synthesis in the presence of human DNA polymerases β, η, ι, and κ. While (S)-N(6)-HB-dA I was readily bypassed by all four enzymes, only polymerases η and κ were able to carry out DNA synthesis past (R,R)-N(6),N(6)-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (S)-N(6)-HB-dA I. In contrast, hPol β was completely blocked by (R,R)-N(6),N(6)-DHB-dA, while hPol η and κ inserted A, G, C, or T opposite the adduct with similar frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed that while translesion synthesis past (S)-N(6)-HB-dA I was mostly error-free, replication of DNA containing (R,R)-N(6),N(6)-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (S)-N(6)-HB-dA I lesions are not miscoding, but that exocyclic (R,R)-N(6),N(6)-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson-Crick pairs with dT.

Research paper thumbnail of Major Groove Orientation of the (2 S )- N 6 -(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine DNA Adduct Induced by 1,2-Epoxy-3-butene

Chemical Research in Toxicology, 2014

1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen... more 1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. It is oxidized by cytochrome P450 monooxygenases to 1,2-epoxy-3butene (EB), which alkylates DNA. BD exposures lead to large numbers of mutations at A:T base pairs even though alkylation of guanines is more prevalent, suggesting that one or more adenine adducts of BD play a role in BD-mediated genotoxicity. However, the etiology of BD-mediated genotoxicity at adenine remains poorly understood. EB alkylates the N 6 exocyclic nitrogen of adenine to form N 6-(hydroxy-3buten-1-yl)-2′-dA ((2S)-N 6-HB-dA) adducts (

Research paper thumbnail of NanoLC/ESI+ HRMS3 Quantitation of DNA Adducts Induced by 1,3-Butadiene

Journal of The American Society for Mass Spectrometry, 2014

Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest f... more Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest fires is of great concern because of its potent carcinogenicity. The adverse health effects of BD are mediated by its epoxide metabolites such as 3,4-epoxy-1-butene (EB), which covalently modify genomic DNA to form promutagenic nucleobase adducts. Because of their direct role in cancer, BD-DNA adducts can be used as mechanism-based biomarkers of BD exposure. In the present work, a mass spectrometry-based methodology was developed for accurate, sensitive, and precise quantification of EB-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) DNA adducts in vivo. In our approach, EB-GII adducts are selectively released from DNA backbone by neutral thermal hydrolysis, followed by ultrafiltration, offline HPLC purification, and isotope dilution nanoLC/ESI(+)-HRMS(3) analysis on an Orbitrap Velos mass spectrometer. Following method validation, EB-GII lesions were quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of EB and in liver tissues of rats exposed to sub-ppm concentrations of BD (0.5-1.5 ppm). EB-GII concentrations increased linearly from 1.15 ± 0.23 to 10.11 ± 0.45 adducts per 10(8) nucleotides in HT1080 cells treated with 0.5-10 μM EB. EB-GII concentrations in DNA of laboratory rats exposed to 0.5, 1.0, and 1.5 ppm BD were 0.17 ± 0.05, 0.33 ± 0.08, and 0.50 ± 0.04 adducts per 10(8) nucleotides, respectively [corrected]. We also used the new method to determine the in vivo half-life of EB-GII adducts in rat liver DNA (2.20 ± 0.12 d) and to detect EB-GII in human blood DNA. To our knowledge, this is the first application of nanoLC/ESI(+)-HRMS(3) Orbitrap methodology to quantitative analysis of DNA adducts in vivo.

Research paper thumbnail of Erratum to: NanoLC/ESI+ HRMS3 Quantitation of DNA Adducts Induced by 1,3-Butadiene

Journal of The American Society for Mass Spectrometry, 2014

in our recently published article contains several errors that were introduced during article pro... more in our recently published article contains several errors that were introduced during article production. The adduct numbers should be expressed "per 10 8 nucleotides" rather than "per 10 6 nucleotides." Also, "0.5-10 μM DEB" should be read "0.5-10 μM EB.

Research paper thumbnail of Synthesis of Site-Specific DNA–Protein Conjugates and Their Effects on DNA Replication

ACS Chemical Biology, 2014

DNA−protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome up... more DNA−protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radicalgenerating systems. As a result of their considerable size and their pronounced effects on DNA−protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts.

Research paper thumbnail of Translesion Synthesis across 1,N6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6- -HMHP-dA) Adducts by Human and Archebacterial DNA Polymerases

Journal of Biological Chemistry, 2012

Background: 1,N 6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2Ј-deoxyadenosine (1,N 6-␥-HMHP-dA) ... more Background: 1,N 6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2Ј-deoxyadenosine (1,N 6-␥-HMHP-dA) adducts are formed in DNA by 1,2,3,4-diepoxybutane (metabolite of human carcinogen 1,3-butadiene). Results: hpols and carry out translesion synthesis, incorporating T, G, or A opposite the 1,N 6-␥-HMHP-dA adduct. Conclusion: Translesion bypass of 1,N 6-␥-HMHP-dA adducts by TLS polymerases is error-prone. Significance: This study identifies 1,N 6-␥-HMHP-dA as the DNA adduct potentially responsible for A3 T and A3 C transversions and deletions induced by 1,3-butadiene. The 1,N 6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2deoxyadenosine (1,N 6-␥-HMHP-dA) adducts are formed upon bifunctional alkylation of adenine nucleobases in DNA by 1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene. The presence of a substituted 1,N 6propano group on 1,N 6-␥-HMHP-dA is expected to block the Watson-Crick base pairing of the adducted adenine with thymine, potentially contributing to mutagenesis. In this study, the enzymology of replication past site-specific 1,N 6-␥-HMHP-dA lesions in the presence of human DNA polymerases (hpols) ␤, , , and and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol , , and Dpo4 were able to copy the modified template. In contrast, hpol inserted a single base opposite 1,N 6-␥-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol ␤. Single nucleotide incorporation experiments indicated that although hpol , , and Dpo4 incorporated the correct nucleotide (dTMP) opposite the lesion, dGMP and dAMP were inserted with a comparable frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed the ability of bypass polymerases to insert dTMP, dAMP, or dGMP opposite 1,N 6-␥-HMHP-dA and detected large amounts of ؊1 and ؊2 deletion products. Taken together, these results indicate that hpol and enzymes bypass 1,N 6-␥-HMHP-dA lesions in an error-prone fashion, potentially contributing to A3 T and A3 C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane.

Research paper thumbnail of Synthesis of DNA oligodeoxynucleotides containing structurally defined N6-(2-hydroxy-3-buten-1-yl)-adenine adducts of 3,4-epoxy-1-butene

Chemico-Biological Interactions, 2007

3,4-Epoxy-1-butene (EB) is generated by cytochrome P450-mediated epoxidation of 1,3-butadiene (BD... more 3,4-Epoxy-1-butene (EB) is generated by cytochrome P450-mediated epoxidation of 1,3-butadiene (BD), an important environmental and industrial chemical classified as a probable human carcinogen. The ability of EB to induce point mutations at GC and AT base pairs has been attributed to its reactions with DNA to form covalent nucleobase adducts. Guanine alkylation is preferred at the endocyclic N7 nitrogen, while adenine can be modified at the N1-, N3-, N7-, and the N 6 positions. For each of these sites, a pair of regioisomeric 2-hydroxy-3-buten-1-yl and 1-hydroxy-3-buten-2-yl adducts is produced as a result of epoxide ring opening at the terminal C-4 or the internal C-3 carbon position of EB, respectively. The N 6-EB-adenine adducts are of particular interest because of their stability in DNA, potentially leading to their accumulation in vivo. In the present work, synthetic DNA oligomers containing structurally defined N 6-(2-hydroxy-3-buten-1-yl)-dA (N 6-HB-dA) adducts were prepared for the first time by a postoligomerization approach that involved coupling 6-chloropurine-containing DNA with synthetic 1-amino-3-buten-2-ol. N 6-HB-dA-containing DNA oligomers were isolated by reversed phase HPLC, and the presence of N 6-HB-dA in their structure was confirmed by molecular weight determination from HPLC-ESI −-MS of the intact strands and by HPLC-ESI +-MS/MS and MS/MS/MS analyses of the enzymatic digests using synthetic N 6-HB-dA as an authentic standard. N 6-HB-dA-containing oligomers generated in this study will be used for structural and biological studies.

Research paper thumbnail of Structures of Exocyclic R,R - and S,S - N 6 , N 6 -(2,3-Dihydroxybutan-1,4-diyl)-2′-Deoxyadenosine Adducts Induced by 1,2,3,4-Diepoxybutane

Chemical Research in Toxicology, 2014

1,3-Butadiene (BD) is an industrial and environmental chemical present in urban air and cigarette... more 1,3-Butadiene (BD) is an industrial and environmental chemical present in urban air and cigarette smoke, and is classified as a human carcinogen. It is oxidized by cytochrome P450 to form 1,2,3,4-diepoxybutane (DEB); DEB bis-alkylates the N 6 position of adenine in DNA. Two enantiomers of bis-N 6-dA adducts of DEB have been

Research paper thumbnail of Structure Elucidation of DNA-Protein Crosslinks by Using Reductive Desulfurization and Liquid Chromatography-Tandem Mass Spectrometry

ChemBioChem, 2014

Easier with ethyl: Guengerich and co-workers have developed a powerful new approach to the struct... more Easier with ethyl: Guengerich and co-workers have developed a powerful new approach to the structure elucidation of hydrolytically stable AGT-DNA crosslinks by reductive desulfurization of the thioether linkage between AGT and DNA to convert cysteine DPCs to the corresponding ethyl-DNA adducts, which can be readily characterized by LC-MSn.

Research paper thumbnail of Synthesis of Sequence-Specific DNA–Protein Conjugates via a Reductive Amination Strategy

Bioconjugate Chemistry, 2013

DNA-protein cross-links (DPCs) are ubiquitous, structurally diverse DNA lesions formed upon expos... more DNA-protein cross-links (DPCs) are ubiquitous, structurally diverse DNA lesions formed upon exposure to bis-electrophiles, transition metals, UV light, and reactive oxygen species. Because of their superbulky, helix distorting nature, DPCs interfere with DNA replication, transcription, and repair, potentially contributing to mutagenesis and carcinogenesis. However, the biological implications of DPC lesions have not been fully elucidated due to the difficulty in generating site-specific DNA substrates representative of DPC lesions formed in vivo. In the present study, a novel approach involving postsynthetic reductive amination has been developed to prepare a range of hydrolytically stable lesions structurally mimicking the DPCs produced between the N7 position of guanine in DNA and basic lysine or arginine side chains of proteins and peptides.

Research paper thumbnail of DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells

Biochemistry, 2013

Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to ... more Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions, including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O(6)-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Smaller numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles.