Transcriptional bypass of regioisomeric ethylated thymidine lesions by T7 RNA polymerase and human RNA polymerase II.

Alkylative damage to DNA can be induced by environmental chemicals, endogenous metabolites and some commonly prescribed chemotherapeutic agents. The regioisomeric N3-, O(2)- and O(4)-ethylthymidine (N3-, O(2)- and O(4)-EtdT, respectively) represent an important class of ethylated DNA lesions. Using nonreplicative double-stranded vectors containing an N3-EtdT, O(2)-EtdT or O(4)-EtdT at a defined site in the template strand, herein we examined the effects of these lesions on DNA transcription mediated by single-subunit T7 RNA polymerase or multisubunit human RNA polymerase II in vitro and in human cells.

A Meta-Analysis on the Relations between EGFR R521K Polymorphism and Risk of Cancer.

The EGFR R521K polymorphism has been shown to reduce the activity of EGFR; however, the association between EGFR R521K polymorphism and the risk of cancer remains inconclusive; therefore we performed a meta-analysis to evaluate the relationship between EGFR R521K polymorphism and susceptibility to cancer. Our results suggest that the EGFR R521K polymorphism is not associated with risk of cancer, but the different chemosensitivity to anticancer drugs may need further investigation.

Effects of Tet-mediated oxidation products of 5-methylcytosine on DNA transcription in vitro and in mammalian cells.

5-methylcytosine (5-mC) is a well-characterized epigenetic regulator in mammals. Recent studies showed that Ten-eleven translocation (Tet) proteins can catalyze the stepwise oxidation of 5-mC to produce 5-hydroxymethylcytosine (5-HmC), 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC). The exciting discovery of these novel cytosine modifications has stimulated substantial research interests about their roles in epigenetic regulation.

Global discovery of protein kinases and other nucleotide-binding proteins by mass spectrometry.

Nucleotide-binding proteins, such as protein kinases, ATPases and GTP-binding proteins, are among the most important families of proteins that are involved in a number of pivotal cellular processes. However, global study of the structure, function, and expression level of nucleotide-binding proteins as well as protein-nucleotide interactions can hardly be achieved with the use of conventional approaches owing to enormous diversity of the nucleotide-binding protein family. Recent advances in mass spectrometry (MS) instrumentation, coupled with a variety of nucleotide-binding protein enrichment methods, rendered MS-based proteomics a powerful tool for the comprehensive characterizations of the nucleotide-binding proteome, especially the kinome.

Profiling global kinome signatures of the radioresistant MCF-7/C6 breast cancer cells using MRM-based targeted proteomics.

Ionizing radiation is widely used in cancer therapy; however, cancer cells often develop radioresistance, which compromises the efficacy of cancer radiation therapy. Quantitative assessment of the alteration of the entire kinome in radioresistant cancer cells relative to their radiosensitive counterparts may provide important knowledge to define the mechanism(s) underlying tumor adaptive radioresistance and uncover novel target(s) for effective prevention and treatment of tumor radioresistance. By employing a scheduled multiple-reaction monitoring analysis in conjunction with isotope-coded ATP affinity probes, we assessed the global kinome of radioresistant MCF-7/C6 cells and their parental MCF-7 human breast cancer cells.

Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells.

Sequential enrichment with titania-coated magnetic mesoporous hollow silica microspheres and zirconium arsenate-modified magnetic nanoparticles for the study of phosphoproteome of HL60 cells.

As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS).

Arsenite binds to the RING finger domains of RNF20-RNF40 histone E3 ubiquitin ligase and inhibits DNA double-strand break repair.

Arsenic is a widespread environmental contaminant. However, the exact molecular mechanisms underlying the carcinogenic effects of arsenic remain incompletely understood. Core histones can be ubiquitinated by RING finger E3 ubiquitin ligases, among which the RNF20-RNF40 heterodimer catalyzes the ubiquitination of histone H2B at lysine 120.

Syntheses and characterizations of the in vivo replicative bypass and mutagenic properties of the minor-groove O2-alkylthymidine lesions.

Endogenous metabolism, environmental exposure, and treatment with some chemotherapeutic agents can all give rise to DNA alkylation, which can occur on the phosphate backbone as well as the ring nitrogen or exocyclic nitrogen and oxygen atoms of nucleobases. Previous studies showed that the minor-groove O(2)-alkylated thymidine (O(2)-alkyldT) lesions are poorly repaired and persist in mammalian tissues. In the present study, we synthesized oligodeoxyribonucleotides harboring seven O(2)-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu or sBu, at a defined site and examined the impact of these lesions on DNA replication in Escherichia coli cells.

Quantitative mass spectrometry-based analysis of β-D-glucosyl-5-hydroxymethyluracil in genomic DNA of Trypanosoma brucei.

β-D-glucosyl-5-hydroxymethyluracil (base J) is a hyper-modified nucleobase found in the nuclear DNA of kinetoplastid parasites. With replacement of a fraction of thymine in DNA, J is localized primarily in telomeric regions of all organisms carrying this modified base. The biosynthesis of J occurs in two putative steps: first, a specific thymine in DNA is recognized and converted into 5-hydroxymethyluracil (5-HmU) by J-binding proteins (JBP1 and JBP2); a glucosyl transferase (GT) subsequently glucosylates the 5-HmU to yield J.

Tet-mediated formation of 5-hydroxymethylcytosine in RNA.

Oxidation of 5-methylcytosine in DNA by ten-eleven translocation (Tet) family of enzymes has been demonstrated to play a significant role in epigenetic regulation in mammals. We found that Tet enzymes also possess the activity of catalyzing the formation of 5-hydroxymethylcytidine (5-hmrC) in RNA in vitro. In addition, the catalytic domains of all three Tet enzymes as well as full-length Tet3 could induce the formation of 5-hmrC in human cells.

Facile enzymatic synthesis of base J-containing oligodeoxyribonucleotides and an analysis of the impact of base J on DNA replication in cells.

We reported here the use of T4 bacteriophage β-glucosyltransferase (T4 β-GT) for the facile synthesis of base J-containing oligodeoxyribonucleotides (ODNs). We found that the enzyme could catalyze the glucosylation of 5-hydroxymethyl-2-deoxyuridine (5hmU) in both single- and double-stranded ODNs, though the latter reaction occurred only when 5hmU was mispaired with a guanine. In addition, base J blocked moderately DNA replication, but it did not induce mutations during replication in human cells.

Effects of tet-induced oxidation products of 5-methylcytosine on DNA replication in mammalian cells.

Recently 5-hydroxymethyl-2'-deoxycytidine (5hmdC), 5-formyl-2'-deoxycytidine (5fdC), and 5-carboxyl-2'-deoxycytidine (5cadC) were discovered in mammalian DNA as oxidation products of 5-methyl-2'-deoxycytidine (5mdC) induced by the ten-eleven translocation family of enzymes. These oxidized derivatives of 5mdC may not only act as intermediates of active cytosine demethylation in mammals but also serve as epigenetic marks on their own. It remains unclear how 5hmdC, 5fdC, and 5cadC affect DNA replication in mammalian cells.

Identification of the glucosyltransferase that converts hydroxymethyluracil to base J in the trypanosomatid genome.

O-linked glucosylation of thymine in DNA (base J) is an important regulatory epigenetic mark in trypanosomatids. β-d-glucopyranosyloxymethyluracil (base J) synthesis is initiated by the JBP1/2 enzymes that hydroxylate thymine, forming 5-hydroxymethyluracil (hmU). hmU is then glucosylated by a previously unknown glucosyltransferase.

Photoswitchable formation of a DNA interstrand cross-link by a coumarin-modified nucleotide.

A coumarin-modified pyrimidine nucleoside (1) has been synthesized using a Cu(I)-catalyzed click reaction and incorporated into oligodeoxynucleotides (ODNs). Interstrand cross-links are produced upon irradiation of ODNs containing 1 at 350 nm. Cross-linking occurs through a [2+2] cycloaddition reaction with the opposing thymidine, 2'-deoxycytidine, or 2'-deoxyadenosine.

Effects of Tet-induced oxidation products of 5-methylcytosine on Dnmt1- and DNMT3a-mediated cytosine methylation.

We investigated systematically the effects of Tet-induced oxidation products of 5-methylcytosine on Dnmt1- and DNMT3a-mediated cytosine methylation in synthetic duplex DNA. We found that the replacement of 5-methylcytosine at a CpG site with a 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxylcytosine or 5-hydroxymethyluracil resulted in altered methylation of cytosine at both the opposite and the neighboring CpG sites. Our results provided important new knowledge about the implications of the 5-methylcytosine oxidation products in maintenance cytosine methylation.

α-N-methylation of damaged DNA-binding protein 2 (DDB2) and its function in nucleotide excision repair.

DDB2 exhibits a high affinity toward UV-damaged DNA, and it is involved in the initial steps of global genome nucleotide excision repair. Mutations in the DDB2 gene cause the genetic complementation group E of xeroderma pigmentosum, an autosomal recessive disease manifested clinically by hypersensitivity to sunlight exposure and an increased predisposition to skin cancer. Here we found that, in human cells, the initiating methionine residue in DDB2 was removed and that the N-terminal alanine could be methylated on its α-amino group in human cells, with trimethylation being the major form.

Cytotoxic and mutagenic properties of regioisomeric O²-, N3- and O⁴-ethylthymidines in bacterial cells.

Exposure to environmental agents and endogenous metabolism can both give rise to DNA alkylation. Thymine is known to be alkylated at O(2), N3 and O(4) positions; however, it remains poorly explored how the regioisomeric alkylated thymidine lesions compromise the flow of genetic information by perturbing DNA replication in cells. Herein, we assessed the differential recognition of the regioisomeric O(2)-, N3- and O(4)-ethylthymidine (O(2)-, N3- and O(4)-EtdT) by the DNA replication machinery of Escherichia coli cells.

Comprehensive characterization of (S)GTP-binding proteins by orthogonal quantitative (S)GTP-affinity profiling and (S)GTP/GTP competition assays.

Thiopurine drugs are widely used as antileukemic drugs and immunosuppressive agents, and 6-thioguanosine triphosphate ((S)GTP) is a major metabolite for these drugs. Recent studies have suggested that thiopurine drugs may exert their cytotoxic effects partly through binding of (S)GTP to a GTP-binding protein, Rac1. However, it remains unclear whether (S)GTP can also bind to other cellular proteins.

Fragmentation of electrospray-produced deprotonated ions of oligodeoxyribonucleotides containing an alkylated or oxidized thymidine.

Alkylation and oxidation constitute major routes of DNA damage induced by endogenous and exogenous genotoxic agents. Understanding the biological consequences of DNA lesions often necessitates the availability of oligodeoxyribonucleotide (ODN) substrates harboring these lesions, and sensitive and robust methods for validating the identities of these ODNs. Tandem mass spectrometry is well suited for meeting these latter analytical needs.

Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts.

Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic.

A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses.

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination.

Cd²⁺-induced alteration of the global proteome of human skin fibroblast cells.

Cadmium (Cd(2+)) is a toxic heavy metal and a well-known human carcinogen. The toxic effects of Cd(2+) on biological systems are diverse and thought to be exerted through a complex array of mechanisms. Despite the large number of studies aimed to elucidate the toxic mechanisms of action of Cd(2+), few have been targeted toward investigating the ability of Cd(2+) to disrupt multiple cellular pathways simultaneously and the overall cellular responses toward Cd(2+) exposure.

A targeted quantitative proteomics strategy for global kinome profiling of cancer cells and tissues.

Kinases are among the most intensively pursued enzyme superfamilies as targets for anti-cancer drugs. Large data sets on inhibitor potency and selectivity for more than 400 human kinases became available recently, offering the opportunity to design rationally novel kinase-based anti-cancer therapies. However, the expression levels and activities of kinases are highly heterogeneous among different types of cancer and even among different stages of the same cancer.

Epimeric 2-deoxyribose lesions: Products from the improper chemical repair of 2-deoxyribose radicals.

Genomic integrity is constantly challenged by DNA damaging agents such as reactive oxygen species (ROS). Consequently, DNA damage can compromise the fidelity and efficiency of essential DNA metabolic processes, including replication and transcription, which may contribute significantly to the etiology of many human diseases. Here, we review one family of DNA lesions, the epimeric 2-deoxyribose lesions, which arise from the improper chemical repair of the 2-deoxyribose radicals.