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Ni(II)-tetradentate amido-quinoline complexes effectively catalysed C-H chlorination of a series of hydrocarbons in the presence of NaOCl and acetic acid. The bond dissociation energy of the C(sp

Cu(ii)-tetradentate amido-quinoline complexes effectively catalysed aromatic C–H hydroxylation using H2O2 as an oxidant in the absence of an external base with a high selectivity of around 90% for phenols via the non-radical pathway (TON ≥720).

Arene metal precursors on treatment with thiosemicarbazone ligands (L1, L2, and L3) yielded a series of cationic mono- and binuclear complexes (1–9) with N∩S bonding mode. In general, the complexes have been formulated as [(p-cymene)Ru(L)Cl]+ and [Cp*2M2(L)2]4+ where L = L1, L2, L3, M = Rh/Ir. Contrary to previous results [1], ruthenium complexes portrayed mononuclear bidentate chelation while rhodium and iridium complexes revealed dinuclear bridging coordination through sulfur and imine nitrogen atoms. All these complexes have been characterized by various spectroscopic techniques and antibacterial studies have been carried out for the complexes as well as the ligands, where 5 and 8 showed good inhibitory antibacterial activity.

Dropwise condensation can yield heat fluxes up to an order of magnitude higher than filmwise condensation. Coalescence is the primary mode of growth for condensing droplets above a small threshold size (e.g., radius r > 2 μm for water at 1 atm), but no prior studies have quantitatively assessed heat transfer during coalescence. Previous models of dropwise condensation have generally described coalescence as an instantaneous event, with a step reduction in heat transfer rate. However, coalescence and recovery of a quasi-steady droplet temperature profile requires a finite time, during which the direct droplet condensation heat transfer rate gradually decays. Additionally, during this period, the droplet may oscillate, repeatedly clearing the surrounding surface and resulting in high overall heat fluxes. This study employs Volume-of-Fluid (VOF) simulations to quantitatively assess these two transient heat transfer processes during droplet coalescence. It is shown that the direct mechanism of gradual heat transfer decay can be represented by a decaying exponential function with a time constant τ . Simulations are performed to determine τ ( r 1 , Rt ) for ( 1 μ m ⩽ r 1 ⩽ 25 μ m; 1 ⩽ Rt ⩽ 4 ) where r 1 is the radius of the smaller droplet and Rt is the radius ratio between the two merging droplets. For water at atmospheric pressure this spans the range of droplet sizes through which most of the heat transfer occurs on a surface ( ∼ 80 % ). A simple correlation is proposed for τ ( r 1 , Rt ) for the studied droplet size range, fluid properties, and surface conditions. These simulations are also employed to determine the order of magnitude of heat transfer enhancement due to repeated clearing of the surrounding surface as droplets coalesce. Findings can inform improved models of dropwise condensation that more accurately predict transient heat transfer during coalescence events.

Imino-quinolyl Schiff-base ligands have been prepared by the condensation reaction of substituted 2-aminopyridine and quinoline-2-carbaldehyde. The reaction of [(arene)MCl2]2 with imino-quinolyl Schiff-base ligands leads to the formation of cationic complexes [(arene)M(L)Cl]+ (1–12). Single crystal X-ray diffraction studies were used to confirm the coordination mode and structures of these complexes. The molecular structures of these complexes revealed that they adopt characteristic three-legged piano stool geometry with the metal coordinating through a terminal chloride and imino-quinolyl ligands in a bidentate chelating NN′ fashion. The ligand coordinates to the metal center through the nitrogen of the quinoline and the imine nitrogen forming a five-membered metallacycle. These compounds were evaluated for their in vitro antibacterial activity by the agar well diffusion method against Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae strains. Results show that all the ligands and complexes inhibited the growth of bacteria.

“I do not think we would conquer this country, unless we break the very backbone of this nation, which is her spiritual and cultural heritage, and therefore, I propose that we replace her old and ancient education system, her culture, for if the Indians think that all that is foreign and English is good and greater than their own, they will lose their self-esteem, their native culture and they will become what we want them, a truly dominated nation.” - Thomas Babington Macaulay. This paper quotes the ever-controversial Thomas Babington Macaulay at the outset because it seems we have long forgotten the gravity our roots hold. It is, now, imperative that we, the Nepali people realize the gold mine we are sitting on, our unique identity and the heritage associated with it. We have already lost a lot; some in the name of development and some in the crevices of time. Our languages, education system, music, food, buildings, settlements, the uniqueness and practices which are associated with our civilization are the ones that set us apart from the world. While the researchers wholeheartedly agree that certain practices such as Sati, Chaupadi, Deuki, Jhuma were indeed violations of inherent human rights in the name of customs, culture and practices and had to be eradicated, we also equally raise voices for those aspects of our culture that we can celebrate, namely our cultural heritage. Now maybe the right time to decide what we want in the form of development? Another copy of a Western City or a Nepal that is unique in all its rights, a Nepal where road expansion is not synonyms to development, where speaking English is not the yardstick for assessing education and where we make our own rules for development. This paper aims to raise questions regarding the said issues. This paper is based on the doctrines related to cultural heritage in Nepal. The researchers carried out on-site surveillance but owing to the lack of technical expertise in the field, the researchers have based their conclusions and recommendations mainly on the reports of Department of Archaeology, experts in the field of cultural heritage in Nepal and government documents depicting the scenario of heritage in Nepal, pre and post the 2015 earthquake.

Halide bridged arene d6 platinum group metal precursors on treatment with thiourea derivatives (L1 and L2) yielded a series of neutral mono-dentate complexes (1–8). In general complexes have been formulated as [(arene)M(L)к1(S)Cl2] where L = L1, M = Ru, arene = p-cymene 1; benzene 2; arene = Cp*, M = Rh 3 and Ir 4; L = L2, M = Ru, p-cymene 5; benzene 6; arene = Cp*, M = Rh 7 and Ir 8. Structural studies revealed that thiourea ligand coordinate to the metal in a mono-dentate fashion via S atom. Further treatment of mono-dentate complexes 1 and 5 with NaN3 in polar solvent resulted in the formation of highly strained к2(N,S) azido complexes 9 and 10 whereas reaction of complex 7 yielded a six membered ring к2(S,O) azido complex 11. Reaction of complex 9 with dimethylacetylene dicarboxylate (DMAD) and diethylacetylene dicarboxylate (DEAD) leads to the formation of nitrogen (N2) bound triazolato complexes 12 and 13 whereas reaction of complex 11 with the same yielded nitrogen (N1) bound triazolato complexes 14 and 15. However reaction of complex 10 with both DMAD and DEAD leads to decomposition of the products. All these complexes have been characterized by various spectroscopic techniques. The molecular structures of the representative complexes 1, 2, 3, 6, 7, 9, 11 and 12 have been determined by single crystal X-ray diffraction study.

The Friedlander condensation reaction between 2-aminonicotinealdehyde and acetophenone derivatives in presence of potassium hydroxide yielded 2-substituted-1,8-napthyridine derivatives viz. 2-(1,8-napthyridin-2-yl)phenol (PHNp), 2-(1,8-napthyridin-2-yl)aniline (AnNp) and 2-(pyridine-4-yl)-1,8-napthyridine (PyNp). Treatment of the chloro-bridged dimers [(arene)MCl2]2 [arene = p-cymene, Cp*; M = Ru, Rh and Ir] with two equivalents of napthyridine ligands (PHNp, AnNp and PyNp) allowed the formation of mononuclear napthyridine complexes having formula [(arene)M(PHNp)Cl2] (1–3), [(arene)M(AnNp)Cl]PF6 (4–6) and [(arene)M(PyNp)Cl2] (7–9). These napthyridine compounds were isolated as neutral and cationic complexes which were further characterized by analytical and spectroscopic techniques. The molecular structures of some of the respective napthyridine complexes were established by carrying out the single crystal X-ray analysis. Single crystal X-ray studies revealed the coordination of the napthyridine ligands to the metal center wherein AnNp ligand coordinated metal in a bidentate chelating NN′ manner and PHNp and PyNp ligand coordinated metal in a monodentate fashion. In case of PHNp complexes the coordination occurs through napthyridine nitrogen N(1) whereas in case of PyNp complexes the coordination takes place through pyridine nitrogen N(1). These napthyridine complexes possessed cytotoxicity against HCT-116 (human colorectal cancer) and MIA-PaCa-2 (pancreatic carcinoma) cancer cell lines as compared to non-cancer cell line ARPE-19.

Dropwise condensation has been identified as a promising heat transfer mechanism because it can yield heat fluxes up to an order of magnitude higher than typically found in filmwise condensation. Models for dropwise condensation generally assume a statistical distribution of droplet sizes and integrate heat transfer over the droplet size spectrum, considering droplet curvature effects on saturation temperature, conduction thermal resistance, and interfacial resistance. Most earlier studies have assumed a constant heat transfer factor ( f = O (1)) to account for the conduction contribution to total thermal resistance. However, f varies with droplet Biot number (Bi) and contact angle ( θ ). Formulations for f with broad ranges of applicability are not currently available. In this study, finite element simulations are performed to determine f and corresponding numerical uncertainties for 0.0001 ⩽ Bi ⩽ 1000 and 10° ⩽ θ ⩽ 170°. This spans the active droplet size range considered in most droplet condensation studies ( e.g. , for water condensing at P atm on a surface 10 K below the ambient temperature, active droplets have 0.0005 f and is validated with published results. The proposed correlation can facilitate modeling and analysis of dropwise condensation.

The reaction of multidentate azine Schiff-base ligands was investigated towards d 6 half-sandwich metal complexes. Tetradentate azine ligand L1 reacts with [(arene)MCl 2 ] 2 (arene = p -cymene, Cp*; M = Ru, Rh and Ir) in 1:2 or 1:1 M ratio to give mononuclear complexes having formula [(arene)M{L1к 3 ( N,N′,N´´ ) }] 2+ whereas the reaction of one equivalent of [(arene)MCl 2 ] 2 with four fold excess of hexadentate azine ligand L2 afforded mononuclear complexes bearing formula [(arene)M{L2к 2 ( N,N′ ) }] + . The reaction of L2 with [( p -cymene)RuCl 2 ] 2 in 1:1 M ratio gave dinuclear complex [( p -cymene) 2 Ru 2 Cl 2 L2к 4 (N,N′,N´´,N´´´) ] 2+ whereas the reaction of L2 with [Cp*MCl 2 ] 2 yielded two coordination isomers (dinuclear and mononuclear). The coordination isomers were separated by column chromatography and characterized by spectral and structural studies. In mononuclear complexes with ligand L1 it acted as tridentate chelating ligand coordinating metal center in a tridentate к 3 fashion through both the pyridine and one azine nitrogen atom leading to the formation of five and six membered chelated rings. Ligand L2 in mononuclear complexes coordinated metal in a bidentate к 2 mode coordinating through both the pyridine nitrogen's whereas in dinuclear complexes L2 acted as tetradentate bridging ligand coordinating both metal atoms in a bidentate к 2 fashion through pyridine nitrogen's thus forming a six membered metallacycle with both the metal centers. In the other isomer of rhodium and iridium complexes L2 acted as tridentate chelating ligand having bonding properties similar with complexes of ligand L1.

The reaction of multidentate azine Schiff-base ligands was investigated towards p -cymene ruthenium, Cp*Rh and Cp*Ir complexes. The reaction of [(arene)MCl 2 ] 2 (arene = p -cymene, Cp*; M = Ru, Rh and Ir) with azine Schiff-base ligands (L1-L3) in 1:2 M ratio led to the formation of mononuclear complexes of the type [(arene)M(L)Cl] + whereas the reaction of [Cp*MCl 2 ] 2 (M = Rh/Ir) with azine Schiff-base ligands (L1-L3) in 1:1 M ratio afforded dinuclear rhodium and iridium complexes bearing formula [{Cp*MCl} 2 (μ-L)] + (M = Rh/Ir). The ligands in dinuclear complexes exhibited interesting coordination modes towards the metal atom. The reaction of ligand L3 with [Cp*IrCl 2 ] 2 dimer yielded two coordination isomers whereas with [Cp*RhCl 2 ] 2 yielded exclusively one isomer. The molecular structure of both the isomers of the iridium complex has been established. In rhodium and iridium complexes the ligand L3 acted as uninegative pentadentate bridging ligand coordinating one metal center in a tridentate fashion and the other metal in a bidentate fashion. In the other isomer the ligand L3 behaved as uninegative tetradentate bridging ligand coordinating both iridium centers in a bidentate fashion. In the mononuclear complexes, the ligands are coordinated to the metal atom in a bidentate N∩N fashion through pyridine nitrogen and azine nitrogen. The ligand (L1 and L2) acted as tetradentate bridging ligand in dinuclear complexes. All these complexes were isolated and characterized by spectroscopic and analytical techniques.

The reaction of [(p-cymene)RuCl2]2 and [Cp*MCl2]2 (M = Rh/Ir) with chelating ligand 2-pyridyl cyanoxime {pyC(CN)NOH} leads to the formation of neutral oximato complexes having the general formula [(arene)M{pyC(CN)NO}Cl] {arene = p-cymene, M = Ru, (1); Cp*, M = Rh (2); Cp*, M = Ir (3)}. Whereas the reaction of 2-pyridyl phenyloxime {pyC(Ph)NOH} and 2-thiazolyl methyloxime {tzC(Me)NOH} with precursor compounds afforded the cationic oxime complexes bearing formula [(arene)M{pyC(ph)NOH}Cl]+ and [(arene)M{tzC(Me)NOH}Cl]+ {arene = p-cymene M = Ru, (4), (7); Cp*, M = Rh (5), (8); Cp*, M = Ir (6), (9)}. The cationic complexes were isolated as their hexafluorophosphate salts. All these complexes were fully characterized by analytical, spectroscopic and X-ray diffraction studies. The molecular structures of the complexes revealed typical piano stool geometry around the metal center within which the ligand acts as a NN′ donor chelating ligand. The Chemo-sensitivity activities of the complexes evaluated against HT-29 (human colorectal cancer), and MIAPaCa-2 (human pancreatic cancer) cell line showed that the iridium-based complexes are much more potent than the ruthenium and rhodium analogues. Theoretical studies were carried out to have a deeper understanding about the charge distribution pattern and the various electronic transitions occurring in the complexes.

Electronic properties of low dimensional systems are particularly sensitive to surface adsorbates. Clear understanding of such phenomena can lead to highly effective and nondestructive material engineering techniques. In this work, water adsorption at the surface of LaAlO3/SrTiO3 heterostructures is systematically studied. The saturation of surface dangling bonds by spontaneous water chemisorptions is found to be a main enabler of the formation of the interface 2D electron gas. In particular, when imbalanced distributions of water based ions, namely protons and hydroxyls, are generated, interface electron doping or depletion becomes surface adsorbates dominant and independent of the LaAlO3 layer thickness. The investigations also reveal the importance of hydrogen bonding through molecular water layers, which provides an energetically feasible pathway for manipulating the surface-bond protons and thus the interface electrical characteristics.

The reaction of [(arene)MCl2]2 with pyridylpyrazolyl ligands (L1 and L2) in the presence of ammonium hexafluorophosphate leads to formation of cationic complexes having the general formula [(arene)M(L)Cl]PF6 {M = Ru, arene = p-cymene (1, 4); Cp*, M = Rh (2, 5); Cp*, M = Ir (3, 6); L = 2-(1H-pyrazol-1-yl)pyridine (L1), 2-(3,5-dimethyl-1H-pyrazol-1-yl)pyridine (L2)}. Similarly the reaction of [CpRu(PPh3)2Cl] and [(ind)Ru(PPh3)2Cl] (ind = η5-C9H7) with L1 and L2 yielded cationic complexes which have been formulated as [(Cp/ind)Ru(L)PPh3]PF6 (7–10). All these complexes were characterized by analytical and spectroscopic techniques. The pyridylpyrazolyl ligands coordinated metal through pyridyl and pyrazolyl nitrogens forming a six-membered metallacycle. The ligands as well as the complexes were evaluated for their in vitro antibacterial activity by agar well diffusion method against two Gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two Gram positive bacteria (Staphylococcus aureus and Bacillus thuriengiensis). Results show that the ligands and the complexes have significant antibacterial activity against Gram negative bacteria.

The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.

© The Author(s) 2016. Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3©-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowedmeasurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.

Repair of oxidative stress- and inflammation-induced DNA lesions by the base excision repair (BER) pathway prevents mutation, a form of genomic instability which is often observed in cancer as 'mutation hotspots'. This suggests that some sequences have inherent mutability, possibly due to sequence-related differences in repair. This study has explored intrinsic mutability as a consequence of sequence-specific repair of lipid peroxidation-induced DNA adduct, 1, N(6)-ethenoadenine (eA). For the first time, we observed significant delay in repair of ϵA at mutation hotspots in the tumor suppressor gene p53 compared to non-hotspots in live human hepatocytes and endothelial cells using an in-cell real time PCR-based method. In-cell and in vitro mechanism studies revealed that this delay in repair was due to inefficient turnover of N-methylpurine-DNA glycosylase (MPG), which initiates BER of eA. We determined that the product dissociation rate of MPG at the hotspot codons was ≈5-12-fold lower than the non-hotspots, suggesting a previously unknown mechanism for slower repair at mutation hotspots and implicating sequence-related variability of DNA repair efficiency to be responsible for mutation hotspot signatures.

The Long-Evans Cinnamon (LEC) rat is an animal model for Wilson’s disease. This animal is genetically predisposed to copper accumulation in the liver, increased oxidative stress, accumulation of DNA damage, and the spontaneous development of hepatocellular carcinoma. Thus, this animal model is useful for studying the relationship of endogenous DNA damage to spontaneous carcinogenesis. In this study, we have investigated the apurinic/apyrimidinic endonuclease 1 (APE1)-mediated excision repair of endogenous DNA damage, apurinic/apyrimidinic (AP)-sites, which is highly mutagenic and implicated in human cancer. We found that the activity was reduced in the liver extracts from the acute hepatitis period of LEC rats as compared with extracts from the age-matched Long-Evans Agouti rats. The acute hepatitis period had also a heightened oxidative stress condition as assessed by an increase in oxidized glutathione level and loss of enzyme activity of glyceraldehyde 3-phosphate dehydrogenase, a key redox-sensitive protein in cells. Interestingly, the activity reduction was not due to changes in protein expression but apparently by reversible protein oxidation as the addition of reducing agents to extracts of the liver from acute hepatitis period reactivated APE1 activity and thus, confirmed the oxidation-mediated loss of APE1 activity under increased oxidative stress. These findings show for the first time in an animal model that the repair mechanism of AP-sites is impaired by increased oxidative stress in acute hepatitis via redox regulation which contributed to the increased accumulation of mutagenic AP-sites in liver DNA.

We report on design of a multi-color laser set up that allows for high spectral, time and spatial resolution imaging based on second- and third-order optical nonlinearities in soft condensed matter. Two femtosecond optical parametric oscillators (OPOs) are pumped simultaneously to provide intrinsically synchronized pulses at more than a dozen tunable colors across visible and infrared wavelengths. We demonstrate the use of independently tunable OPOs in a variety of imaging modalities. In one useful application, we explore brain tissue in a two-photon absorption fluorescence imaging experiment with near infrared optical pulses (λ ~ 1,070 nm). We also demonstrate second and sum-frequency generation microscopies in different tissues. Results from application of time-resolved, three-color coherent anti-stokes Raman scattering in tissue are presented to demonstrate feasibility of quantitative spectroscopic imaging.

Two dimensional supersonic flows over a sharp flat plate at zero incidence has been investigated numerically. A fictitious curvature is formed due to the development of a boundary layer at the leading edge of the plate. The pressure along the entire surface and at the trailing edge has been computed over a wide range of Mach numbers and two different temperatures. The temperature variation due to dissipation of kinetic energy within the boundary layer has been analysed numerically. Keywords: Computational Fluid Dynamics, Supersonic Flow, Flat Plate, Numerical Investigation.

The reaction of [Cp*MCl2]2 (M = Rh/Ir) with N–N′ azine Schiff-base ligands (L1–L4) leads to the formation of mononuclear cationic half-sandwich complexes having the general formula [Cp*M(L)Cl]+ (1–8), (M = Rh/Ir and L = (2-hydroxy-4-methoxybenzylidene)2-pyridylamidrazone (L1), (2-hydroxybenzylidene)2-pyridylamidrazone (L2), (1-(2-hydroxyphenyl)ethylidene)2-pyridylamidrazone (L3) and (1-phenylethylidene)2-pyridylamidrazone (L4). All these complexes were isolated as their hexafluorophosphate salts and fully characterized by spectroscopic and analytical techniques. The molecular structure of complexes (1), (3), (4), (7) and (8) have been determined by single crystal X-ray crystallographic studies which displayed the coordination of the ligand to the metal in a bidentate N∩N fashion through nitrogen atom of pyridine and one azine nitrogen. The chemo-sensitivity activities of the complexes were evaluated against HT-29 (human colorectal cancer) cell line and non-cancer cell line ARPE-19 (human retinal epithelial cells) which revealed that the complexes are moderately cytotoxic to cancer cells over human cells although complex 5 was the most potent among all the compounds. Theoretical studies carried out using DFT and TD-DFT at B3LYP level shows good agreement with the experimental results.

DNA topoisomerase 2 (Top2) poisons, including common anticancer drugs etoposide and doxorubicin kill cancer cells by stabilizing covalent Top2-tyrosyl-DNA 5'-phosphodiester adducts and DNA double-strand breaks (DSBs). Proteolytic degradation of the covalently attached Top2 leaves a 5'-tyrosylated blocked termini which is removed by tyrosyl DNA phosphodiesterase 2 (TDP2), prior to DSB repair through non homologous end joining (NHEJ). Thus, TDP2 confers resistance of tumor cells to Top2-poisons by repairing such covalent DNA-protein adducts, and its pharmacological inhibition could enhance the efficacy of Top2-poisons. We discovered NSC111041, a selective inhibitor of TDP2, by optimizing a high throughput screening (HTS) assay for TDP2's 5'-tyrosyl phosphodiesterase activity and subsequent validation studies. We found that NSC111041 inhibits TDP2's binding to DNA without getting intercalated into DNA and enhanced etoposide's cytotoxicity synergistically in TDP2-expressing cells but not in TDP2 depleted cells. Furthermore, NSC111041 enhanced formation of etoposide-induced gamma-H2AX foci presumably by affecting DSB repair. Immuno-histochemical analysis showed higher TDP2 expression in a sub-set of different type of tumor tissues. These findings underscore the feasibility of clinical use of suitable TDP2 inhibitors in adjuvant therapy with Top2-poisons for a sub-set of cancer patients with high TDP2 expression. (C) 2016 Elsevier B.V. All rights reserved.

We report an oxygen surface adsorbates induced metal–insulator transition at the LaAlO3/SrTiO3 interfaces. The observed effects were attributed to the terminations of surface Al sites and the resultant electron-accepting surface states. By controlling the local oxygen adsorptions, we successfully demonstrated the nondestructive patterning of the interface two-dimensional electron gas (2DEG). The obtained 2DEG structures are stable in air and also robust against general solvent treatments. This study provides new insights into the metal–insulator transition mechanism at the complex oxide interfaces and also a highly efficient technique for tailoring the interface properties.

Mammalian apurinic/apyrimidinic endonuclease (APE1) initiates the repair of abasic sites (AP-sites), which are highly toxic, mutagenic, and implicated in carcinogenesis. Also, reducing the activity of APE1 protein in cancer cells and tumors sensitizes mammalian tumor cells to a variety of laboratory and clinical chemotherapeutic agents. In general, mouse models are used in studies of basic mechanisms of carcinogenesis, as well as pre-clinical studies before transitioning into humans. Human APE1 (hAPE1) has previously been cloned, expressed, and extensively characterized. However, the knowledge regarding the characterization of mouse APE1 (mAPE1) is very limited. Here we have expressed and purified full-length hAPE1 and mAPE1 in and from E. coli to near homogeneity. mAPE1 showed comparable fast reaction kinetics to its human counterpart. Steady-state enzyme kinetics showed an apparent K(m) of 91 nM and k(cat) of 4.2 s(-1) of mAPE1 for the THF cleavage reaction. For hAPE1 apparent K(m) and k(cat) were 82 nM and 3.2 s(-1), respectively, under similar reaction conditions. However, k(cat)/K(m) were in similar range for both APE1s. The optimum pH was in the range of 7.5-8 for both APE1s and had an optimal activity at 50-100 mM KCl, and they showed Mg(2+) dependence and abrogation of activity at high salt. Circular dichroism spectroscopy revealed that increasing the Mg(2+) concentration altered the ratio of "turns" to "β-strands" for both proteins, and this change may be associated with the conformational changes required to achieve an active state. Overall, compared to hAPE1, mAPE1 has higher K(m) and k(cat) values. However, overall results from this study suggest that human and mouse APE1s have mostly similar biochemical and biophysical properties. Thus, the conclusions of mouse studies to elucidate APE1 biology and its role in carcinogenesis may be extrapolated to apply to human biology. This includes the development and validation of effective APE1 inhibitors as chemosensitizers in clinical studies.

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5′-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3′- and 5′-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3′- and 5′-phosphotyrosyl linkage at the 3′ and 5′ ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p -nitrophenyl-thymidine-5′-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5′ activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC 50 = 40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.

Chronic inflammation and oxidative stress are arguably associated with an increased risk of cancer. Certain diseases that are characterized by oxyradical overload, such as Wilson's disease (WD), have also been associated with a higher risk of liver cancer. The Long-Evans Cinnamon (LEC) rat, an animal model for WD, is genetically predisposed to the spontaneous development of liver cancer and has been shown to be very useful for studying the mechanisms of inflammation-mediated spontaneous carcinogenesis. Endonuclease III (Nth1) plays a significant role in the removal of oxidative DNA damage. Nth1 and a tumor suppressor gene Tuberous sclerosis 2 (Tsc2) are bi-directionally regulated in humans, mice, and rats by a common minimal promoter containing two Ets-binding sites (EBSs). In this study, we examined the expression of Nth1 and Tsc2 genes during disease progression in the LEC rat liver. During the period of acute hepatitis (16-17 weeks), we observed decreased Nth1 and Tsc2 mRNA levels and a continued decrease of the Tsc2 gene in 24 weeks in LEC rats, while the effect was minimal in Long-Evans Agouti (LEA) rats. This reduction in the mRNA levels was due to the reduced binding of EBSs in the Nth1/Tsc2 promoter. Increase in protein oxidation (carbonyl content) during the same time period (16-24 weeks) may have an effect on the promoter binding of regulatory proteins and consequent decrease in Nth1 and Tsc2 gene expressions during tumorigenesis.

Bolometric detectors based on epitaxial thin films of rare earth perovskite manganites have been proposed as total energy monitors for X-ray pulses at the Linac Coherent Light Source free electron laser. We demonstrate such a detector scheme based on epitaxial thin films of the perovskite manganese oxide material Nd{sub 0.67}Sr{sub x0.33}MnO{sub 3}, grown by pulsed laser deposition on buffered silicon substrates. The substrate and sensor materials are chosen to meet the conflicting requirements of radiation hardness, sensitivity, speed and linearity over a dynamic range of three orders of magnitude. The key challenge in the material development is the integration of the sensor material with Si. Si is required to withstand the free electron laser pulse impact and to achieve a readout speed three orders of magnitude faster than conventional cryoradiometers for compatibility with the Linac Coherent Light Source pulse rate. We discuss sensor material development and the photoresponse of prototype devices. This Linac Coherent Light Source total energy monitor represents the first practical application of manganite materials as bolometric sensors.

In both bacteria and eukaryotes the alkylated, oxidized, and deaminated bases and depurinated lesions are primarily repaired via an endogenous preventive pathway, i.e. base excision repair (BER). Radiation therapy and chemotherapy are two important modes of cancer treatment. Many of those therapeutic agents used in the clinic have the ability to induce the DNA damage; however, they may also be highly cytotoxic, causing peripheral toxicity and secondary cancer as adverse side effects. In addition, the damage produced by the therapeutic agents can often be repaired by the BER proteins, which in effect confers therapeutic resistance. Efficient inhibition of a particular BER protein(s) may increase the efficacy of current chemotherapeutic regimes, which minimizes resistance and ultimately decreases the possibility of the aforementioned negative side effects. Therefore, pharmacological inhibition of DNA damage repair pathways may be explored as a useful strategy to enhance chemosensitivity. Various agents have shown excellent results in preclinical studies in combination chemotherapy. Early phase clinical trials are now being carried out using DNA repair inhibitors targeting enzymes such as PARP, DNA-PK or MGMT. In the case of BER proteins, elimination of N-Methylpurine DNA glycosylase (MPG) or inhibition of AP-endonuclease (APE) increased sensitivity of cancer cells to alkylating chemotherapeutics. MPG(-/-) embryonic stem cells and cells having MPG knock-down by siRNA are hypersensitive to alkylating agents, whereas inhibition of APE by small molecule inhibitors sensitized cancer cells to alkylating chemotherapeutics. Thus, MPG and other BER proteins could be potential targets for chemosensitization.

Human apurinic/apyrimidinic (AP) endonuclease (hAPE) initiates the repair of an abasic site (AP site). To gain insight into the mechanisms of damage recognition of hAPE, we conducted surface plasmon resonance spectroscopy to study the thermodynamics and kinetics of its interaction with substrate DNA containing an abasic site (AP DNA). The affinity of hAPE binding toward DNA increased as much as 6-fold after replacing a single adenine (equilibrium dissociation constant, K(D), 5.3 nm) with an AP site (K(D), 0.87 nm). The enzyme-substrate complex formation appears to be thermodynamically stabilized and favored by a large change in Gibbs free energy, DeltaG degrees (-50 kJ/mol). The latter is supported by a high negative change in enthalpy, DeltaH degrees (-43 kJ/mol) and also positive change in entropy, DeltaS degrees (24 J/(K mol)), and thus the binding process is spontaneous at all temperatures. Analysis of kinetic parameters reveals small enthalpy of activation for association, DeltaH degrees++(ass) (-17 kJ/mol), and activation energy for association (E(a), -14 kJ/mol) when compared with the enthalpy of activation for dissociation, DeltaH degrees++(diss) (26 kJ/mol), and activation energy in the reverse direction (E(d), 28 kJ/mol). Furthermore, varying concentration of KCl showed an increase in binding affinity at low concentration but complete abrogation of the binding at higher concentration, implying the importance of hydrophobic, but predominantly ionic, forces in the Michaelis-Menten complex formation. Thus, low activation energy and the enthalpy of activation, which are perhaps a result of dipole-dipole interactions, play critical roles in AP site binding of APE.

Interest in the mechanisms of DNA repair pathways, including the base excision repair (BER) pathway specifically, has heightened since these pathways have been shown to modulate important aspects of human disease. Modulation of the expression or activity of a particular BER enzyme, N-methylpurine DNA glycosylase (MPG), has been demonstrated to play a role in carcinogenesis and resistance to chemotherapy as well as neurodegenerative diseases, which has intensified the focus on studying MPG-related mechanisms of repair. A specific small molecule inhibitor for MPG activity would be a valuable biochemical tool for understanding these repair mechanisms. By screening several small molecule chemical libraries, we identified a natural polyphenolic compound, morin hydrate, which inhibits MPG activity specifically (IC50 = 2.6 µM). Detailed mechanism analysis showed that morin hydrate inhibited substrate DNA binding of MPG, and eventually the enzymatic activity of MPG. Computational docking studies with an x-ray derived MPG structure as well as comparison studies with other structurally-related flavanoids offer a rationale for the inhibitory activity of morin hydrate observed. The results of this study suggest that the morin hydrate could be an effective tool for studying MPG function and it is possible that morin hydrate and its derivatives could be utilized in future studies focused on the role of MPG in human disease.

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. MPG activity and other DNA glycosylases do not have an absolute requirement for a cofactor. In contrast, all downstream activities of major base excision repair proteins, such as apurinic/apyrimidinic endonuclease, DNA polymerase beta, and ligases, require Mg(2+). Here we have demonstrated that Mg(2+) can be significantly inhibitory toward MPG activity depending on its concentration but independent of substrate type. The pre-steady-state kinetics suggests that Mg(2+) at high but physiologic concentrations decreases the amount of active enzyme concentrations. Steady-state inhibition kinetics showed that Mg(2+) affected K(m), but not V(max), and the inhibition could be reversed by EDTA but not by DNA. At low concentration, Mg(2+) stimulated the enzyme activity only with hypoxanthine but not ethenoadenine. Real-time binding experiments using surface plasmon resonance spectroscopy showed that the pronounced inhibition of activity was due to inhibition in substrate binding. Nonetheless, the glycosidic bond cleavage step was not affected. These results altogether suggest that Mg(2+) inhibits MPG activity by abrogating substrate binding. Because Mg(2+) is an absolute requirement for the downstream activities of the major base excision repair enzymes, it may act as a regulator for the base excision repair pathway for efficient and balanced repair of damaged bases, which are often less toxic and/or mutagenic than their subsequent repair product intermediates.

Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1,N(6)-ethenoadenine, hypoxanthine, and alkylation adducts in DNA. Genetic studies discovered at least eight validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations that result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine residues, Arg-141 and Arg-120, are important for the activity of hMPG as the germ line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared with the WT hMPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (KD) of hMPG toward 1,N(6)-ethenoadenine-containing oligonucleotide (ϵA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple turnover condition. Surface plasmon resonance spectroscopy further showed that both variants had significantly decreased nonspecific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between ϵA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence.

e20511 Background: Mutations in KRAS and NRAS have been found in a number of malignancies, particularly metastatic colorectal cancer, lung adenocarcinoma, and thyroid cancer. Several studies have shown that specific mutations in KRAS and NRAS are less likely to respond to anti-EGFR therapies. Several diagnostics have been developed to identify such mutations. However, in a large number of cancer patients, tumor biopsy poses a significant health risk thereby limiting the tissue available for current molecular diagnostics. Non-invasive liquid biopsy diagnostics are revolutionizing cancer treatment selection, patient prognosis, and monitoring. Low abundance circulating tumor DNA (ctDNA) is purified from patient plasma and used as an alternative source of genomic material for detecting tumor mutations. Currently available qPCR kits for detecting RAS mutations have a limited sensitivity and coverage since they are not optimized for liquid biopsy purposes. Methods: We have developed a two-step PCR to qPCR method to identify down to single mutant copies of KRAS or NRAS in ctDNA isolated from plasma. The mutant copies are first enriched by a novel multiplexed formula of primers and wildtype (WT) allele blockers. The mutations are then identified by EntroGen’s RAS Mutation Screening Panel or next generation sequencing (NGS). Synthetic mutant DNA was spiked in a background of WT DNA purified from plasma to yield a range of mutant allelic burden over WT background. The dilutions were analyzed to determine the method’s sensitivity. We subsequently used the method to screen 53 non-small cell lung cancer (NSCLC) patient samples. Results: Our two-step method detects single digit mutant copies of 35 clinically significant KRAS and NRAS mutations. Furthermore, NGS experiments demonstrated the two-step method can enrich mutations more than 52-fold. KRAS 12/13 mutations were identified in 6 out of 53 (11%) NSCLC patients. Conclusions: ctDNA-based liquid biopsy tests have been employed by few laboratories but are generally available only as a service. EntroGen’s platform agnostic liquid biopsy RAS assay provides clinicians the tools necessary to determine cancer treatment and monitor patients using most PCR and qPCR instruments.

Precise information on dispersion of the nonlinear optical susceptibility of Raman active media is essential in order to get an insight into physics and chemistry of intra- and inter-molecular interactions. We propose and experimentally demonstrate a method that is capable of resolving both real and imaginary parts of third-order nonlinearity (χ (3) ) in the vicinity of Raman resonances. Dispersion of χ (3) can be obtained from a medium probed within microscopic volumes with a spectral resolution of better than 0.1 cm -1 thus making our approach an essential tool in quantitative microscopic characterization of complex biological media. Time-domain CARS transients traced with femtosecond pulses within orders of magnitude in the signal decay can lead to resolution of fine spectral features in χ (3) dispersion that can not be reliably detected by frequency-domain Raman based spectroscopy/microscopy techniques, including coherent methods. We will present results of the method’s application in biological cells and tissue. Namely, we accessed a protein line at 1245 cm -1 in E-coli cell, major DNA and protein lines in red blood cells and triglyceride Raman active peaks in fat tissue.

Half‐sandwich ruthenium, rhodium and iridium complexes (1–12) were synthesised with aldoxime (L1), ketoxime (L2) and amidoxime (L3) ligands. Ligands have the general formula [PyC(R)NOH], where R = H (L1), R = CH3 (L2) and R = NH2 (L3). Reaction of [{(arene)MCl2}2] (arene = p‐cymene, benzene, Cp*; M = Ru, Rh, Ir) with ligands L1–L3 in 1:2 metal precursor‐to‐ligand ratio yielded complexes such as [{(arena)MLκ2(N∩N)Cl}]PF6. All the ligands act as bidentate chelating nitrogen donors in κ2 (N∩N) fashion while forming complexes. In vitro anti‐tumour activity of complexes 2 and 10 against HT‐29 (human colorectal cancer), BE (human colorectal cancer) and MIA PaCa‐2 (human pancreatic cancer) cell lines and non‐cancer cell line ARPE‐19 (human retinal epithelial cells) revealed a comparable activity although complex 2 demonstrated greater selectivity for MIA PaCa‐2 cells than cisplatin. Further studies demonstrated that complexes 3, 6, 9 and 12 induced significant apoptosis in Dalton's ascites lymphoma (DL) cells. In vivo anti‐tumour activity of complex 2 on DL‐bearing mice revealed a statistically significant anti tumour activity (P = 0.0052). Complexes 1–12 exhibit HOMO–LUMO energy gaps from 3.31 to 3.68 eV. Time‐dependent density functional theory calculations explain the nature of electronic transitions and were in good agreement with experiments.

The topoisomerase II (topo II) DNA incision-and-ligation cycle can be poisoned (for example following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double-strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5'-phosphotyrosyl-linked topo II-DNA adducts. Here, X-ray structures of mouse Tdp2-DNA complexes reveal that Tdp2 β-2-helix-β DNA damage-binding 'grasp', helical 'cap' and DNA lesion-binding elements fuse to form an elongated protein-DNA conjugate substrate-interaction groove. The Tdp2 DNA-binding surface is highly tailored for engagement of 5'-adducted single-stranded DNA ends and restricts nonspecific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analyses support a single-metal ion catalytic mechanism for the exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily and establish a molecular framework for targeted small-molecule blockade of Tdp2-mediated resistance to anticancer topoisomerase drugs.

Background Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg2+ in its activity was not studied in sufficient details. Results In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg2+/Mn2+, and that neither Zn2+ or nor Ca2+ can activate hTDP2. Mg2+ also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg2+ controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg2+ concentration above which it is inhibitory for hTDP2 activity. Conclusion These results altogether reveal the optimal Mg2+ requirement in hTDP2 mediated reaction.

Protein purification is still very empirical, and a unified method for purifying proteins without an affinity tag is not available yet. In the postgenomic era, functional genomics, however, strongly demands such a method. In this paper we have formulated a unique method that can be applied for purifying any recombinant basic protein from Escherichia coli. Here, we have found that if the pH of the buffer is merely one pH unit below the isoelectric point (pI) of the recombinant proteins, most of the latter bind to the column. This result supports the Henderson-Hasselbalch principle. Considering that E. coli proteins are mostly acidic, and based on the pI determined theoretically, apparently all recombinant basic proteins (at least pI-1 > or = 6.94) may be purified from E. coli in a single step using a cation-exchanger resin, SP-Sepharose, and a selected buffer pH, depending on the pI of the recombinant protein. Approximately, two-fifths of human proteome, including many if not all nucleic acid-interacting proteins, have a pI of 7.94 or higher; virtually all these 12,000 proteins may be purified using this method in a single step.

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. In this study, we tested the role of excised base on MPG enzymatic activity. After the reaction, MPG produced two products: free damaged base and AP-site containing DNA. Our results showed that MPG excises 1,N(6)-ethenoadenine (varepsilonA) from varepsilonA-containing oligonucleotide (varepsilonA-DNA) at a similar or slightly increased efficiency than it does hypoxanthine (Hx) from Hx-containing oligonucleotide (Hx-DNA) under similar conditions. Real-time binding experiments by surface plasmon resonance (SPR) spectroscopy suggested that both the substrate DNAs have a similar equilibrium binding constant (K(D)) towards MPG, but under single-turnover (STO) condition there is apparently no effect on catalytic chemistry; however, the turnover of the enzyme under multiple-turnover (MTO) condition is higher for varepsilonA-DNA than it is for Hx-DNA. Real-time binding experiments by SPR spectroscopy further showed that the dissociation of MPG from its product, AP-site containing DNA, is faster than the overall turnover of either Hx- or varepsilonA-DNA reaction. We thereby conclude that the excised base plays a critical role in product inhibition and, hence, is essential for MPG glycosylase activity. Thus, the results provide the first evidence that the excised base rather than AP-site could be rate-limiting for DNA-glycosylase reactions.

N -Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N -alkylpurine adducts, deaminated and lipid peroxidation-induced purine adducts. MPG from human and mouse has previously been cloned and expressed. However, due to the poor expression level in Escherichia coli ( E. coli ) and multi-step purification process of full-length MPG, most successful attempts have been limited by extremely poor yield and stability. Here, we have optimized the codons within the first five residues of human MPG (hMPG) to the best used codons for E. coli and expressed full-length hMPG in large amounts. This high expression level in conjunction with a strikingly high isoelectric point (9.65) of hMPG, in fact, helped purify the enzyme in a single step. A previously well-characterized monoclonal antibody having an epitope in the N-terminal tail could detect this codon-optimized hMPG protein. Surface plasmon resonance studies showed an equilibrium binding constant ( K D ) of 0.25 nM. Steady-state enzyme kinetics showed an apparent K m of 5.3 nM and k cat of 0.2 min −1 of MPG for the hypoxanthine (Hx) cleavage reaction. Moreover, hMPG had an optimal activity at pH 7.5 and 100 mM KCl. Unlike the previous reports by others, this newly purified full-length hMPG is appreciably stable at high temperature, such as 50 °C. Thus, this study indicates that this improved expression and purification system will facilitate large scale production and purification of a stable human MPG protein for further biochemical, biophysical and structure–function analysis.

1,N6-Ethenoadenine (epsilonA) is generated endogenously by lipid peroxidation and exogenously by tumorigenic industrial agents, vinyl chloride, and vinyl carbamate. epsilonA detected in human tissues causes mutation and is implicated in liver, colon and lung cancers. N-methyl purine DNA-glycosylase (MPG) is the only enzyme known so far to repair epsilonA. However, the mechanism of in vivo repair of epsilonA and the role of MPG remain enigmatic. Moreover, previous in vivo repair studies for DNA lesions, including epsilonA, focused only on the step of the removal of the base lesion without further insight into the completion of the repair process. This may be in part due to the unavailability of an appropriate in vivo quantitative method to evaluate complete BER process at the basal level. Our newly developed in vivo method is highly sensitive and involves phagemid M13mp18, containing epsilonA at a defined position. The complete repair events have been estimated by plaque assay in E. coli with the phagemids recovered from the human cells after cellular processing. We found that the detectable complete (removal and replacement of epsilonA with adenine) repair was observed only 18% in 16 h, but with the repair nearing completion within 24 h in colon cancer, HCT-116, cells. Moreover, MPG is the predominant enzyme for the BER process to remove epsilonA in mammalian cells. Although, the epsilonA is fairly a bulky adduct compared to other small BER substrate lesions, NER pathway is not involved in repair of this adduct. Furthermore, the epsilonA repair in vivo and in vitro is predominant in the G0/G1 phase of the cell cycle.

N-Methylpurine DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. In this study we tested the role of N-terminal extension on MPG hypoxanthine (Hx) cleavage activity. Our results showed that MPG lacking N-terminal extension excises hypoxanthine with significantly reduced efficiency, one-third of that exhibited by full-length MPG under similar conditions. Steady-state kinetics showed full-length MPG has higher V(max) and lower K(m) than NDelta100 MPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that truncation can substantially increase the equilibrium binding constant of MPG toward Hx, but under single-turnover conditions there is apparently no effect on catalytic chemistry; however, the truncation of the N-terminal tail affected the turnover of the enzyme significantly under multiple turnover conditions. Real time binding experiments by surface plasmon resonance spectroscopy further showed that NDelta100 MPG binds approximately six times more tightly toward its product apurinic/apyrimidinic site than the substrate, whereas full-length MPG similarly binds to both the substrate and the product. We thereby conclude that the N-terminal tail in MPG plays a critical role in overcoming the product inhibition, which is achieved by reducing the differences of MPG binding affinity toward Hx and apurinic/apyrimidinic sites and thus is essential for the Hx cleavage reaction of MPG. The results from this study also affirm the need for reinvestigation of full-length MPG for its enzymatic and structural properties, which are currently available mostly for the truncated protein.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, induced by alkylating chemotherapeutics, and deaminated and lipid peroxidation-induced purine adducts. We have generated monoclonal antibodies (moAbs) against human MPG. Twelve independent hybridoma clones were characterized, which, except 520-16A, are identical based on epitope exclusion assay. Four moAbs, including 520-2A, 520-3A, 520-16A, and 520-26A, have high affinity (K(D) approximately 0.3-1.6nM), and their subtypes were IgG(2a), IgG(1), IgG(2a), and IgG(2b), respectively. moAb 520-3A recognizes the sequence (52)AQAPCPRERCLGPP(66)T, an epitope exclusively present in the N-terminal extension of human MPG. We found that moAb 520-3A significantly inhibited MPG's enzymatic activity towards different substrates, such as hypoxanthine, 1,N(6)ethenoadenine and methylated bases, which represent different classes of DNA damage, however, with different efficiencies. Real-time binding experiments using surface plasmon resonance (SPR) spectroscopy showed that the pronounced inhibition of activity was not in the substrate-binding step. Single turnover kinetics (STO) revealed that the inhibition was at the catalytic step. Since we found that this antibody has an epitope in the N-terminal tail, the latter appears to have an important role in substrate discrimination, however, with a differential effect on different substrates.