Inhibition Of DNA Transcription Research Articles

The Influence of the Auxiliary Ligand in Monofunctional Pt(II) Anticancer Complexes on the DNA Backbone.

Monofunctional platinum complexes offer a promising alternative to cisplatin in cancer chemotherapy, showing a unique mechanism of action. Their ability to induce minor helix distortions effectively inhibits DNA transcription. In our study, we synthesized and characterized three monofunctional Pt(II) complexes with the general formula [Pt(en)(L)Cl]NO3, where en = ethylenediamine, and L = pyridine (py), 2-methylpyridine (2-mepy), and 2-phenylpyridine (2-phpy). The hydrolysis rates of [Pt(en)(py)Cl]NO3 (1) and [Pt(en)(2-mepy)Cl]NO3 (2) decrease with the bulkiness of the auxiliary ligand with k(1) = 2.28 ± 0.15 × 10-4 s-1 and k(2) = 8.69 ± 0.98 × 10-5 s-1 at 298 K. The complex [Pt(en)(2-phpy)Cl]Cl (3) demonstrated distinct behavior. Upon hydrolysis, an equilibrium (Keq = 0.385 mM) between the complexes [Pt(en)(2-phpy)Cl]+ and [Pt(en)(2-phpy-H+)]+ was observed with no evidence (NMR or HR-ESI-MS) for the presence of the aquated complex [Pt(en)(2-phpy)(H2O)]2+. Despite the kinetic similarities between phenanthriplatin and (2), complexes (1) and (2) exhibit minimal activity against A549 lung cancer cell line (IC50 > 100 μΜ), whereas complex (3) exhibits notable cytotoxicity (IC50 = 41.11 ± 2.1 μΜ). In examining the DNA binding of (1) and (2) to the DNA model guanosine (guo), we validated their binding through guoN7, which led to an increased population of the C3'-endo sugar conformation, as expected. However, we observed that the rapid transition 2E (C2'-endo) ↔ 3E (C3'-endo), in the case of [Pt(en)(py)(guo)](NO3)2 ([1-guo]), slows down in the case of [Pt(en)(2-mepy)(guo)](NO3)2 ([2-guo]), resulting in separate signals for the two conformers in the 1H NMR spectra. This phenomenon arises from the steric hindrance between the methyl group of pyridine and the sugar moiety of guanosine. Notably, this hindrance is absent in [2-(9-MeG)] (9-MeG = 9-methylguanine), probably due to the absence of a bulky sugar unit in 9-MeG. In the case of (3), where the bulkiness of the substitution on the pyridine is further increased by a phenyl group, we observed a notable proximity between 9-MeGH8 and the phenyl ring of 2-phpy. Considering that only (3) exhibited good cytotoxicity against the A549 cancer cell line, it is suggested that auxiliary ligands, L, with an extended aromatic system and proper orientation in complexes of the type cis-[Pt(en)(L)Cl]NO3, may enhance the cytotoxic activity of such complexes.

Abstract 1720: 53BP1 regulates heterochromatin through liquid-liquid phase separation (LLPS)

Abstract As compacted DNA, heterochromatin represses abnormal gene expression by inhibiting DNA transcription and maintains genome integrity by protecting aberrant chromosome segregation. Therefore, abnormalities in heterochromatin function are commonly found in cancer. However, how exactly heterochromatin preserves the genome integrity remains poorly understood due largely to the complex structure of heterochromatin. Recently, increasing evidence suggests that heterochromatin factors, including HP1α, KAP1, and SUV39H1/2, could drive the soluble heterochromatin into phase-separated droplets/condensates to promote gene silencing, which is referred to as liquid-liquid phase separation (LLPS). TP53-binding protein 1 (53BP1) has traditionally been a critical factor in DNA double-strand break (DSB) repair. We recently reported an unexpected finding that under normal growth conditions, 53BP1 regulates heterochromatin function through LLPS, which is independent of its DSB repair function. Our studies reveal a new paradigm in studying 53BP1, heterochromatin, and genome stability. However, an important question remains unanswered: how 53BP1 is recruited to heterochromatin to undergo LLPS and the associated broad biological impact. To address this question, we performed a special crosslinking coupled mass spectrometry to uncover potential factors in the 53BP1 condensates assembled on heterochromatin. We identified many factors associated with DNA methylation and histone modification, which are the two main steps in restoring heterochromatin epigenetic modification. Their interactions with 53BP1 at heterochromatin were confirmed by different approaches. We further focused on one of the key factors, PCNA, and carried out a detailed analysis of the interaction between PCNA and 53BP1 and the role of such interaction in 53BP1 LLPS, DSB repair, and heterochromatin function. These findings reveal the previously uncharacterized function of 53BP1 at heterochromatin during normal DNA replication. Citation Format: Xinran Geng, Youwei Zhang. 53BP1 regulates heterochromatin through liquid-liquid phase separation (LLPS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1720.

USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells

Cisplatin is a chemotherapy drug that causes a plethora of DNA lesions and inhibits DNA transcription and replication, resulting in the induction of apoptosis in cancer cells. However, over time, patients develop resistance to cisplatin due to repeated treatment and thus the treatment efficacy is limited. Therefore, identifying an alternative therapeutic strategy combining cisplatin treatment along with targeting factors that drive cisplatin resistance is needed. CRISPR/Cas9 system-based genome-wide screening for the deubiquitinating enzyme (DUB) subfamily identified USP28 as a potential DUB that governs cisplatin resistance. USP28 regulates the protein level of microtubule-associated serine/threonine kinase 1 (MAST1), a common kinase whose expression is elevated in several cisplatin-resistant cancer cells. The expression level and protein turnover of MAST1 is a major factor driving cisplatin resistance in many cancer types. Here we report that the USP28 interacts and extends the half-life of MAST1 protein by its deubiquitinating activity. The expression pattern of USP28 and MAST1 showed a positive correlation across a panel of tested cancer cell lines and human clinical tissues. Additionally, CRISPR/Cas9-mediated gene knockout of USP28 in A549 and NCI-H1299 cells blocked MAST1-driven cisplatin resistance, resulting in suppressed cell proliferation, colony formation ability, migration and invasion in vitro. Finally, loss of USP28 destabilized MAST1 protein and attenuated tumor growth by sensitizing cells to cisplatin treatment in mouse xenograft model. We envision that targeting the USP28-MAST1 axis along with cisplatin treatment might be an alternative therapeutic strategy to overcome cisplatin resistance in cancer patients.

Synthesis and characterization of novel Ru(III) complexes of 2‐aminopyrazine: Interaction with biomolecules, antineoplastic activity, and computational investigation

Ruthenium (III) complexes (1–3) of 2‐aminopyrazine (pyz) with general formula of [Hpyz][RuCl4(DMSO)(pyz)](1), Na[RuCl4(pyz)(DMSO)] (2), and (Hpyz)[RuCl4(pyz)2].2H2O (3) have been synthesized and characterized by elemental analyses, FTIR, 1H NMR, and UV–visible spectroscopy, along with the magnetic susceptibility and cyclic voltammetry measurements. The molecular structures of the complexes have also been optimized using density functional theory (DFT) calculation which demonstrates an octahedral geometry to be adopted by the Ru(III) ion. The UV–visible and fluorescence spectra were employed to study the interaction of the compounds with nucleic acid (ctDNA and tRNA) and bovine serum albumin (BSA). The data showed a higher tendency for the ligand and its complexes (1–3) to interact with biomolecules (1 > 2 > 3). All complexes showed potent in vitro anticancer activity against three human cancer cell lines and high safety against normal cell lines as complex (1) is the most active one, it was selected for the flow cytometric evaluation for cell death mode, cell cycle analysis, and matrix metalloproteinase‐9 (MMP9) expression in treated MDA‐231 cells. Proliferating cell nuclear antigen (PCNA) expression and VEGF concentration were evaluated in the treated cells and compared with the untreated ones. Our study proved that complex (1) arrests the cell cycle, inhibits DNA transcription, reduces both MMP9 (validated by our molecular docking investigation targeting MMP9 protein) and PCNA expressions, and induces apoptotic cell death, leading to cancer metastasis prevention.

Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.

Cas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) effector proteins and show that they provide protection against bacteriophages and plasmids through the targeted DNA binding rather than DNA cleavage. Biochemical assays suggest that Cas12m effectors can act as roadblocks inhibiting DNA transcription and/or replication, thereby triggering interference against invaders. Cryo-EM structure of Gordonia otitidis (Go) Cas12m ternary complex provided here reveals the structural mechanism of DNA binding ensuring interference. Harnessing GoCas12m innate ability to bind DNA target we fused it with adenine deaminase TadA-8e and showed an efficient A-to-G editing in Escherichia coli and human cells. Overall, this study expands our understanding of the functionally diverse Cas12 protein family, revealing DNA-binding dependent interference mechanism of Cas12m effectors that could be harnessed for engineering of compact base-editing tools.

The antifungal spectrum of medicinal plants: A review

Medicinal plant possessed antifungal effects by many mechanisms, they caused membrane disturbance resulting in the loss of membrane integrity, inhibited DNA transcription and reduced the cell populations, inhibited the activity of fungal antioxidant enzymes and inhibited fungal biofilm formation. The current review discussed the antifungal effects of medicinal plants.

Vitamin D Receptor and Retinoid X Receptor Alpha in Melanocytic Benign Lesions and Melanoma.

Vitamin D receptor (VDR) exerts its biological effects when it heterodimerizes to a nuclear receptor of the retinoid family called retinoid X receptor α (RXRα), stimulating or inhibiting DNA transcription. VDR stimulation by vitamin D analogs led to in vitro antiproliferative effects, and experimental RXRα knockout led to loss of proliferation control in melanoma cells. The aim of this study was to determine VDR and RXRα positivity in melanocytic lesions, compared with normal skin species. By immunohistochemistry assays, nuclear VDR, cytoplasmic VDR, and RXRα and RXRα in keratinocytes surrounding melanocytes were evaluated in 77 controls, 92 intradermal nevi, 54 dysplastic nevi, and 83 melanomas in this retrospective cross-sectional study. Nuclear VDR, cytoplasmic VDR, and RXRα were less expressed in exposed areas ( P < 0.001, P = 0.0006, and P < 0.001, respectively) than covered areas. All melanocytic lesions had loss of VDR and RXRα comparing with the control group. In the melanoma group, nuclear VDR tended to inversely correlate with the Breslow index (r = -0.11, P = 0.29) but directly correlated with histological regression ( P = 0.0293). RXRα inversely correlated with mitosis (r = -0.245; P = 0.0263). We can suggest that sun exposure affected VDR and RXRα immunopositivity. Nuclear VDR tendency of inverse correlation with the Breslow index showed that worse melanomas have a greater loss of VDR. RXRα inversely correlated with mitosis, indicating that RXRα can have a role in proliferation control. VDR and RXRα may participate in the development of melanocytic lesions and be a future target of new studies and directed therapies.

Next-Generation Sequencing-Based Analysis of the Effects of N1- and N6-Methyldeoxyadenosine Adducts on DNA Transcription.

DNA methylation can occur naturally or be induced by various environmental and chemotherapeutic agents. The regioisomeric N1- and N6-methyldeoxyadenosine (1mdA and 6mdA, respectively) represent an important class of methylated DNA adducts. In this study, we developed a shuttle vector- and next-generation sequencing-based assay to quantitatively assess the effects of 1mdA and 6mdA on the accuracy and efficiency of DNA transcription. Our results revealed that 1mdA can induce multiple types of mutant transcripts and strongly inhibit DNA transcription, whereas 6mdA is a nonmutagenic DNA adduct that can exhibit a subtle but significant inhibitory effect on DNA transcription in vitro and in human cells. Moreover, our results demonstrated that the transcription-coupled nucleotide excision repair pathway is dispensable for the removal of 1mdA and 6mdA from the template DNA strand in human cells. These findings provided new important insights into the functional interplay between DNA methylation modifications and transcription in mammalian cells.

161 Epigenetic Changes in Poultry Due to Reprogramming of the Gut Microbiota at an Early Stage of Embryonic Development

Abstract Epigenetic regulation of the gene expression is an interaction of the external environment with the genetic information. They are potentially heritable changes in the gene expression which does not involve alteration in DNA sequence and can be triggered by microRNA activity and DNA methylation. MicroRNA is fraction of small RNA molecules that have a fundamental impact on gene expression. DNA methylation inhibits DNA transcription by addition of the methyl residues to the cysteine within the CpG islands of the gene promoters. These processes can be modulated by environmental factors, such as intestinal microbiota modification. In poultry, the microbiota can be reprogrammed using in ovo technology at an early stage of embryo development. The intestinal microbiota is therefore stimulated and rearranged by injecting bioactive substances into air chamber of eggs on the day 12 of incubation. We have proved that the administration of lactic acid bacteria strains and galactooligosaccharide in ovo is effective in modulating of the intestinal microbiota. The administration of bioactive compounds has been demonstrated to influence gene expression in immune, intestinal and metabolic tissues. However, it has been noticed that a significant part of genes is silenced. In our experiment after in ovo administration of the substances in different genotypes (chicken broiler and native Polish breed) the range of tissues was collected: liver, caecal tonsils, spleen. By performing the bioinformatic analysis of the expression microarray, silenced genes and active miRNAs were selected. Methylation was analysed using the global and MSP-qPCR method, and analysis of miRNA activity using miRCURY LNA PCR Systems. We confirmed that negative regulation of the gene expression have epigenetic character and its mechanism depends on the genotype and the substance administered in ovo. Epigenetic nature of research is new direction of host-microbiome interaction. Research was financed by grant UMO-2017/25/N/NZ9/01822 funded by National Science Centre (Poland).

Strategic approach of multifaceted antibacterial mechanism of limonene traced in Escherichia coli

Antibacterial potential of Limonene against Multi Drug Resistant (MDR) pathogens was studied and mechanism explored. Microscopic techniques viz. Fluorescent Microscopy (FM), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) indicated membrane disruption, cellular leakage and cell death of Escherichia coli (E. coli) cells when treated with limonene. Leakage of intracellular proteins, lipids and nucleic acid confirmed membrane damage and disruption of cell permeability barrier. Further, release of intracellular ATP, also suggested disruption of membrane barrier. Interaction of limonene with DNA revealed its capability in unwinding of plasmid, which could eventually inhibit DNA transcription and translation. Differential expression of various proteins and enzymes involved in transport, respiration, metabolism, chemotaxis, protein synthesis confirmed the mechanistic role of limonene on their functions. Limonene thus can be a potential candidate in drug development.

Hepatic DNA Methylation in Response to Early Stimulation of Microbiota with Lactobacillus Synbiotics in Broiler Chickens

DNA methylation inhibits DNA transcription by the addition of methyl residues to cysteine within the CpG islands of gene promoters. The process of DNA methylation can be modulated by environmental factors such as intestinal microbiota. In poultry, the composition of the intestinal microbiota can be stimulated by in ovo delivery of synbiotics. The present study aims to determine the effect of Lactobacillus synbiotics delivered in ovo on the level of hepatic DNA methylation in broiler chickens. In ovo stimulation was performed on day 12 of egg incubation. Bioactive compounds delivered in ovo included (S1)—Lactobacillus salivarius with GOS and (S2)—Lactobacillus plantarum with RFO. Samples were collected from six individuals from each group on day 42 post-hatching. DNA methylation of five genes selected on the basis of the transcriptome data were analyzed using the qMSP method. Significant changes were observed in DNA methylation of genes in liver including ANGPTL4 and NR4A3, after S2 delivery. The obtained results confirm that the downregulation of metabolic gene expression in the liver mediated by in ovo stimulation had epigenetic characteristics.

DNA Binding three Azo Dyes as new Antibiotics

The antimicrobial activities of (E)-4-((2-nitrophenyl) diazenyl) benzene-1,3-diol (1), (E)-4-((3-nitrophenyl) diazenyl) benzene-1,3-diol (2) and (E)-4-((4-nitrophenyl) diazenyl) benzene-1,3-diol (3), were screened in vitro towered Staphylococcus aureus strain NCTC 6571 and Candida Krusei. The results were revealed that the azo dye (2), which has NO2 group in the meta-position, was more active towered Staphylococcus aureus using 0.3 mg / mL and 0.4 mg/ mL concentrations. This activity was seemed to be higher than the activity of (1) and (3), which have substituted NO2 group in ortho-and para-positions respectively. However, the azo dye (1) was gave well activity against Candida Krusei than (2) and (3) using same concentrations. These results were then compared with that obtained by antibiotics (erythromycin capsules, amoxicillin capsules and metheprim tablet). The results were displayed that the activities of different concentrations of each azo dye were better than the antibiotics in the treatment of the disseminated infection. Further, the three azo dyes were showed non-toxic effects toward of the hemolytic red blood cells and didn’t show any hemolysis effect in the cells. Further the three azo dyes were variable in their bonded to the infected human DNA. The azo dyes (1) and (3) can affect the human DNA resulting DNA damage, which inhibits DNA transcription and replication. However, the azo dye (2) was right bind the human DNA without damaged it in contrast with the control. Due to recommend the three synthetic azo dyes as new antibiotics for disseminated infection. The first section in your paper

Tissue Expression of Prohibition-I and Its Relationship With Prognostic Factors in Breast Cancer

Globally, breast cancer is the most common malignancy among females. Prohibition (PHB)-I, a homologous protein, was initially introduced as a suppressor gene for amplification process. Further, the protein has a key role in the cell cycle and is capable of inhibiting DNA transcription in many cell types. Therefore, its possible role in different types of human malignancies is of interest.The current study aimed at examining the relationship between the tissue distribution of PHB-I and prognostic factors of breast cancer. Paraffin-embedded tissue specimens of 33 patients diagnosed with breast cancer at Omid teaching Hospital, Mashhad, Iran were studied and a commercial monoclonal antibody was used to perform immunohistochemistry (IHC). The relationship between PHB-I tissue expression with age, disease stage, tumor grade and size, as well as hormone receptor status including estrogen (ER) and progesterone (PR) receptors, and Her-2 receptor were evaluated. The Immunohistochemical analysis showed a relative increase in PHB-I tissue expression along with higher tumor grade (P=0.057). In addition, higher expression of ER and PR were observed (P=0.027 and 0.009, respectively). The age of patients and other prognostic factors including Her-2 receptor status and disease stage did not statistically correlate with PHB-I expression. An increased expression of PHB-I was observed in the breast cancer tumors of the current study patients compared with the anatomically healthy margin. Its coloration with some prognostic factors such as disease grade and expression of ER and PR might indicate the PHB-I potential application for diagnostic and patient management purposes.

In vitro antitumor activity of novel benzimidazole-based Cu(II) complexes

Four benzimidazole-based Cu(II) complexes: Cu2(p-2-bmp)2Br4 (1), Cu2(p-2-bmp)2Cl4 (2), Cu2(p-2-bmb)2(DMF)2Br4·(CHCl3) (3), and Cu(p-2-bmb)(NO3)2·(CHCl3) (4) were isolated and characterized, where p-2-bmp is 1-((2-(pyridine-2-yl)-1H-benzoimidazol-1-yl)methyl)-1H-pyridine and p-2-bmb is 1-((2-(pyridin-2-yl)-1-benzoimidazol-1-yl)methyl)-1H-benzotriazole. Complexes 1 and 2 have binuclear configurations, 3 has a mononuclear structure, and 4has a one-dimensional (1-D) chain skeleton. To evaluate their potential anticancer effects on human carcinoma cells, anti-proliferation, DNA binding and cleavage, and apoptosis elicitation were examined. Compared with complexes 2, 3, and 4, complex 1 exhibited potent in vitro cytotoxicity toward four cell lines (MCF7, EC109, SH-SY5Y and QBC939), with SH-SY5Y cells demonstrating the most sensitivity. Therefore, further in-depth investigations were performed using complex 1. Absorption titration experiments, circular dichroism spectroscopic studies, and ethidium bromide displacement assays suggested that complex 1 binds to DNA through intercalation, significantly cleaves supercoiled pBR322 DNA, and inhibits DNA transcription. Cell cycle analysis revealed that SH-SY5Y cells were arrested in the G2/M phase after treatment with complex 1. Membrane permeability analysis and nuclear staining of SH-SY5Y cells showed that complex 1 could induce apoptosis.

What's in a Name?

Nomenclature is important—it codifies terms on which members of a guild mutually concur, so as to understand one another better instead of rambling along in imprecise babble. In this issue, Jeffrey Flier and Joseph Loscalzo have thoughtfully applied this principle to how certain sectors of our profession are labeled. They have been resourceful in their analysis and balanced in their recommendations. Names and terms do mean something, usually for the better. But sometimes, they need interpretation. When I first heard about “translational research” many years ago, I was excited, because having worked on RNA and gene expression, I thought it meant that more NIH dollars would be flowing to the ribosome and related fields. Alas. I had two encounters with nomenclature in my first days as an independent investigator. Shortly after publication of one of the first papers from my laboratory, I received a letter (an actual letter, in an envelope, with a stamp) from one Waldo E. Cohn at Oak Ridge National Laboratory, telling me that “SDS” was an unacceptable term for the detergent I had put into my electrophoretic gels. (For more than you've ever wanted to know about the advent of SDS gel electrophoresis, see ref. 1.) Dr. Cohn said that he was in charge of biochemical nomenclature and told me it had to be written as “NaDodSO4.” Having been an undergraduate student of chemistry, where its elegant nomenclature was one of the things that had turned me on, I thought this term was totally nuts from every angle, chemistry or etymology. A couple of years later, a copyeditor at Academic Press told me that I could not use the term “dry ice” in an article but that I had to say “solid CO2.” I retorted that some of my colleagues might not even know that solid CO2 meant dry ice, but I lost the battle and again needed the publication. (It also occurred to me on that occasion, for the first time, that had life used its chemical sibling silicon instead of carbon, we would be sitting on dry ice on the beach with lungs that could handle sand. I give that copyeditor credit for this idea.) Nomenclature issues are not the same as ambiguity or mispronunciation, the latter as when an innocent student says that he has inhibited DNA transcription with the antibiotic “actomyosin,” or simple grammatical errors (2), such as when one says that he has changed “the media” on one plate of cells. And at a deeper level, nomenclature and definition often feud. A “stem cell” can be defined in several ways, and each way of thinking about such cells has subtle nuances of differentiation (no pun intended). So, I say that thinking about nomenclature and being careful are important, and thus, thank Flier and Loscalzo. What they present is one way to avoid having the biomedical profession descend into “alt-terms,” with the resulting danger of being taken as “alt-facts,” for how we name our scientific corridors.

606 - A novel role for Top2α as a mediator of resistance to ribosomal DNA transcription inhibition

606 - A novel role for Top2α as a mediator of resistance to ribosomal DNA transcription inhibition

Downregulation of β-Adrenoceptors in Isoproterenol-Induced Cardiac Remodeling through HuR.

β-adrenergic receptors (β-ARs) play an important role in cardiac remodeling, which is the key pathological process in various heart diseases and leads to heart failure. However, the regulation of β-AR expression in remodeling hearts is still unclear. This study aims to clarify the possible mechanisms underlying the regulation of β1- and β2-AR expression in cardiac remodeling. The rat model of cardiac remodeling was established by subcutaneous injection of isoproterenol(ISO) at the dose of 0.25 mg·kg−1·d−1 for 7days. We found that the expression of β1- and β2-ARs decreased in the remodeling heart. The mechanisms may include the inhibition of DNA transcription and the increase of mRNA degradation. cAMP-response element binding protein(CREB) is a well-known transcription factor of β-AR. However, the expression and activation of CREB was not changed in the remodeling heart. Further, human Antigen-R (HuR), a RNA binding protein, which binds to the 3'-untranslated region of the β-AR mRNA and promotes RNA degradation, was increased in the remodeling model. And in vitro, HuR deficiency reversed the reduction of β-AR mRNA induced by ISO. Therefore, the present findings indicate that HuR, but not CREB, is responsible for the reduction of β-AR expression in ISO induced cardiac remodeling.

Mass Spectrometry-Based Quantitative Strategies for Assessing the Biological Consequences and Repair of DNA Adducts.

The genetic integrity of living organisms is constantly threatened by environmental and endogenous sources of DNA damaging agents that can induce a plethora of chemically modified DNA lesions. Unrepaired DNA lesions may elicit cytotoxic and mutagenic effects and contribute to the development of human diseases including cancer and neurodegeneration. Understanding the deleterious outcomes of DNA damage necessitates the investigation about the effects of DNA adducts on the efficiency and fidelity of DNA replication and transcription. Conventional methods for measuring lesion-induced replicative or transcriptional alterations often require time-consuming colony screening and DNA sequencing procedures. Recently, a series of mass spectrometry (MS)-based strategies have been developed in our laboratory as an efficient platform for qualitative and quantitative analyses of the changes in genetic information induced by DNA adducts during DNA replication and transcription. During the past few years, we have successfully used these MS-based methods for assessing the replicative or transcriptional blocking and miscoding properties of more than 30 distinct DNA adducts. When combined with genetic manipulation, these methods have also been successfully employed for revealing the roles of various DNA repair proteins or translesion synthesis DNA polymerases (Pols) in modulating the adverse effects of DNA lesions on transcription or replication in mammalian and bacterial cells. For instance, we found that Escherichia coli Pol IV and its mammalian ortholog (i.e., Pol κ) are required for error-free bypass of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) in cells. We also found that the N(2)-CEdG lesions strongly inhibit DNA transcription and they are repaired by transcription-coupled nucleotide excision repair in mammalian cells. In this Account, we focus on the development of MS-based approaches for determining the effects of DNA adducts on DNA replication and transcription, where liquid chromatography-tandem mass spectrometry is employed for the identification, and sometimes quantification, of the progeny products arising from the replication or transcription of lesion-bearing DNA substrates in vitro and in mammalian cells. We also highlight their applications to lesion bypass, mutagenesis, and repair studies of three representative types of DNA lesions, that is, the methylglyoxal-induced N(2)-CEdG, oxidatively induced 8,5'-cyclopurine-2'-deoxynucleosides, and regioisomeric alkylated thymidine lesions. Specially, we discuss the similar and distinct effects of the minor-groove DNA lesions including N(2)-CEdG and O(2)-alkylated thymidine lesions, as well as the major-groove O(4)-alkylated thymidine lesions on DNA replication and transcription machinery. For example, we found that the addition of an alkyl group to the O(4) position of thymine may facilitate its preferential pairing with guanine and thus induce exclusively the misincorporation of guanine nucleotide opposite the lesion, whereas alkylation of thymine at the O(2) position may render the nucleobase unfavorable in pairing with any of the canonical nucleobases and thus exhibit promiscuous miscoding properties during DNA replication and transcription. The MS-based strategies described herein should be generally applicable for quantitative measurement of the biological consequences and repair of other DNA lesions in vitro and in cells.

DNA studies of newly synthesized heteroleptic platinum(II) complexes [Pt(bpy)(iip)](2+) and [Pt(bpy)(miip)](2.).

Two new mono-nuclear heteroleptic platinum(II) complexes, [Pt(bpy)(iip)](PF6)2 (1) and [Pt(bpy)(miip)](PF6)2·2H2O (2) (bpy is 2,2'-bipyridine; iip is 2-(imidazo-4-yl)-1H-imidazo[4,5-f] [1,10] phenanthroline; miip is 2-(1-methylimidazo-2-yl)-1H-imidazo[4,5-f] [1, 10] phenanthroline), have been synthesized and fully characterized by CHN analysis, electrospray ionization and MALDI-TOF mass spectrometry, (1)H NMR, FT-IR (ATR), and UV-Vis spectrophotometer. Cytotoxicity, ability to inhibit DNA transcription and DNAse activity of the complexes were studied. The DNA-binding behaviors of both complexes have also been studied by spectroscopic methods, cyclic voltammetry and viscosity measurements. Both complexes showed cytotoxic properties and 2 was more cytotoxic than 1. DNA transcription was inhibited upon increasing concentrations of both complexes. The complex 2 was found to be a better inhibitor than 1. The same pattern can be seen in the DNAse profile of the complexes. In addition, 2 was found to promote cleavage of pBR322 DNA at a lower concentration than 1. The spectroscopic, electrochemical and viscometric results indicate that both complexes show some degree of binding to DNA in an intercalative mode, resulting in intrinsic binding constants K b = 3.55 ± 0.6 × 10(4) M(-1) and 7.01 ± 0.9 × 10(4) M(-1) for 1 and 2, respectively. The difference in the DNA-binding affinities of 1 and 2 may presumably be explained by the methylated imidazole nitrogen atom that makes the compound more hydrophobic and gives better intercalative binding ability to DNA's hydrophobic environment.

Transcriptional inhibition and mutagenesis induced by N-nitroso compound-derived carboxymethylated thymidine adducts in DNA.

N-nitroso compounds represent a common type of environmental and endogenous DNA-damaging agents. After metabolic activation, many N-nitroso compounds are converted into a diazoacetate intermediate that can react with nucleobases to give carboxymethylated DNA adducts such as N3-carboxymethylthymidine (N3-CMdT) and O4-carboxymethylthymidine (O4-CMdT). In this study, we constructed non-replicative plasmids carrying a single N3-CMdT or O4-CMdT, site-specifically positioned in the transcribed strand, to investigate how these lesions compromise the flow of genetic information during transcription. Our results revealed that both N3-CMdT and O4-CMdT substantially inhibited DNA transcription mediated by T7 RNA polymerase or human RNA polymerase II in vitro and in human cells. In addition, we found that N3-CMdT and O4-CMdT were miscoding lesions and predominantly directed the misinsertion of uridine and guanosine, respectively. Our results also suggested that these carboxymethylated thymidine lesions may constitute efficient substrates for transcription-coupled nucleotide excision repair in human cells. These findings provided important new insights into the biological consequences of the carboxymethylated DNA lesions in living cells.