Crystal structure, DNA binding studies, nucleolytic property and topoisomerase I inhibition of zinc complex with 1,10-phenanthroline and 3-methyl-picolinic acid

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Factors affecting nucleolytic efficiency of some ternary metal complexes with DNA binding and recognition domains. Crystal and molecular structure of Zn(phen)(edda)

The binding selectivity of the M(phen)(edda) (M = Cu, Co, Ni, Zn; phen = 1,10-phenanthroline, edda = ethylenediaminediacetic acid) complexes towards ds(CG)(6), ds(AT)(6) and ds(CGCGAATTCGCG) B-form oligonucleotide duplexes were studied by CD spectroscopy and molecular modeling. The binding mode is intercalation and there is selectivity towards AT-sequence and stacking preference for A/A parallel or diagonal adjacent base steps in their intercalation. The nucleolytic properties of these complexes were investigated and the factors affecting the extent of cleavage were determined to be: concentration of complex, the nature of metal(11) ion, type of buffer, pH of buffer, incubation time, incubation temperature, and the presence of hydrogen peroxide or ascorbic acid as exogenous reagents. The fluorescence property of these complexes and its origin were also investigated. The crystal structure of the Zn(phen)(edda) complex is reported in which the zinc atom displays a distorted trans-N4O2 octahedral geometry; the crystal packing features double layers of complex molecules held together by extensive hydrogen bonding that inter-digitate with adjacent double layers via pi...pi interactions between 1,10-phenanthroline residues. The structure is compared with that of the recently described copper(II) analogue and, with the latter, included in molecular modeling. (C) 2008 Elsevier B.V. All rights reserved.

LaTBP1: A Leishmania amazonensis DNA-binding protein that associates in vivo with telomeres and GT-rich DNA using a Myb-like domain

Different species of Leishmania can cause a variety of medically important diseases, whose control and treatment are still health problems. Telomere binding proteins (TBPs) have potential as targets for anti-parasitic chemotherapy because of their importance for genome stability and cell viability. Here, we describe LaTBP1 a protein that has a Myb-like DNA-binding domain, a feature shared by most double-stranded telomeric proteins. Binding assays using full-length and truncated LaTBP1 combined with spectroscopy analysis were used to map the boundaries of the Myb-like domain near to the protein only tryptophan residue. The Myb-like domain of LaTBP1 contains a conserved hydrophobic cavity implicated in DNA-binding activity. A hypothetical model helped to visualize that it shares structural homology with domains of other Myb-containing proteins. Competition assays and chromatin immunoprecipitation confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs, suggesting that LaTBP1 is a new TBP. (C) 2007 Elsevier B.V. All rights reserved.

The Influence of Solutes on the Enthalpy/Entropy Change of the Actinomycin D Binding to DNA: Hydration, Energy Compensation and Long-Range Deformation on DNA

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Synthesis, cytotoxic activity and DNA interaction of Pd(II) complexes bearing N′-methyl-3,5-dimethyl-1-thiocarbamoylpyrazole

A new series of complexes of general formulae [PdX2(tmdmPz)] {X = Cl (1), Br (2), I (3), SCN (4); tmdmPz = N′-methyl-3,5-dimethyl-1- thiocarbamoylpyrazole} have been synthesized and characterized by elemental analysis, molar conductivities, IR, 1H and 13C{ 1H} NMR spectroscopy. In these complexes, the tmdmPz coordinates to Pd(II) center as a neutral N,S-chelating ligand. The geometries of the complexes have been optimized with the DFT method. Cytotoxicity evaluation against LM3 (mammary adenocarcinoma) and LP07 (lung adenocarcinoma) cell lines indicated that complexes 1-4 were more active than cisplatin. The binding of the complexes with a purine base (guanosine) was investigated by 1H NMR and mass spectrometry, showing that the coordination of guanosine occurs through N7. Electrophoretic DNA migration studies showed that all of them modify the DNA tertiary structure. © 2013 Elsevier Ltd. All rights reserved.

The bacterial chromosome segregation protein Spo0J spreads along DNA from parS nucleation sites

Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres. (C) 2007 Elsevier B.V. All rights reserved.

DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Leishmania amazonensis TRF ( TTAGGG repeat-binding factor) homologue binds and co-localizes with telomeres

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A systematic approach to identify STRE-binding proteins of the gsn glycogen synthase gene promoter in Neurospora crassa

The gene encoding glycogen synthase in Neurospora crassa (gsn) is transcriptionally down-regulated when mycelium is exposed to a heat shock from 30 to 45 degrees C. The gsn promoter has one stress response element (STRE) motif that is specifically bound by heat shock activated nuclear proteins. In this work, we used biochemical approaches together with mass spectrometric analysis to identify the proteins that bind to the STRE motif and could participate in the gsn transcription regulation during heat shock. Crude nuclear extract of heat-shocked mycelium was prepared and fractionated by affinity chromatography. The fractions exhibiting DNA-binding activity were identified by electrophoretic mobility shift assay (EMSA) using as probe a DNA fragment containing the STRE motif DNA-protein binding activity was confirmed by Southwestern analysis. The molecular mass (MM) of proteins was estimated by fractionating the crude nuclear extract by SDS-PAGE followed by EMSA analysis of the proteins corresponding to different MM intervals. Binding activity was detected at the 30-50 MM kDa interval. Fractionation of the crude nuclear proteins by IEF followed by EMSA analysis led to the identification of two active fractions belonging to the pIs intervals 3.54-4.08 and 6.77-7.31. The proteins comprising the MM and pI intervals previously identified were excised from a 2-DE gel, and subjected to mass spectrometric analysis (MALDI-TOF/TOF) after tryptic digestion. The proteins were identified by search against the MIPS and MIT N. crassa databases and five promising candidates were identified. Their structural characteristics and putative roles in the gsn transcription regulation are discussed.

Polymorphisms of DNA repair genes XRCC1 and XRCC3, interaction with environmental exposure and risk of chronic gastritis and grastic cancer

Aim: To evaluate the association between polymorphisms XRCC1 Arg194Trp and Arg399Gln and XRCC3 Thr241Met and the risk for chronic gastritis and gastric cancer, in a Southeastern Brazilian population. Methods: Genotyping by PCR-RFLP was carried out on 202 patients with chronic gastritis (CG) and 160 patients with gastric cancer (GC), matched to 202 (C1) and 150 (C2) controls, respectively. Results: No differences were observed among the studied groups with regard to the genotype distribution of XRCC1 codons 194 and 399 and of XRCC3 codon 241. However, the combined analyses of the three variant alleles (194Trp, 399Gln and 241Met) showed an increased risk for chronic gastritis when compared to the GC group. Moreover, an interaction between the polymorphic alleles and demographic and environmental factors was observed in the CG and GC groups. XRCC1 194Trp was associated with smoking in the CG group, while the variant alleles XRCC1 399Gln and XRCC3 241Met were related with gender, smoking, drinking and H pylori infection in the CG and GC groups. Conclusion: Our results showed no evidence of a rela-tionship between the polymorphisms XRCC1 Arg194Trp and Arg399Gln and XRCC3 Thr241Met and the risk of chronic gastritis and gastric cancer in the Brazilian population, but the combined effect of these variants may interact to increase the risk for chronic gastritis, considered a premalignant lesion. Our data also indicate a gene-environment interaction in the susceptibility to chronic gastritis and gastric cancer. © 2005 The WJG Press and Elsevier Inc. All rights reserved.

DNA repair gene polymorphism is associated with the genetic basis of atherosclerotic coronary artery disease

Background: Atherosclerotic coronary artery disease (CAD) is a multifactorial process that appears to be caused by the interaction of environmental risk factors with multiple predisposing genes. It is nowadays accepted that increased levels of DNA damage induced by xenobiotics play an important role in the early phases of atherogenesis. Therefore, in this study, we focus on determining whether genetic variations in xenobiotic-metabolizing [glutathione-S-transferase theta 1 (GSTT1), glutathione-S-transferase mu 1 (GSTM1), cytochrome P450 IIEI (CYP2E1)] and DNA repair [X-ray cross-complementing group 1 (XRCC1)] genes might be associated with increased risk for CAD. Methods: A case-control study was conducted with 400 individuals who underwent subjected to coronary angiography. A total of 299 were patients diagnosed with effective coronary atherosclerosis (case group; >20% obstructive lesion), and 101 (control group) were individuals diagnosed as negative for CAD (<20% obstructive lesions). The polymorphism identifications for GSTM1 and GSTT1, and for CYP2E1 and XRCC1 genes were performed by polymerase chain reaction (PCR) amplification and by PCR-RFLP, respectively. Results and conclusions: The XRCC1 homozygous wild-type genotype Arg/Arg for codon 399 was statistically less pronounced in the case subjects (21.4%) than in controls (38.5%); individuals with the variant XRCC1 genotype had a 2.3-fold increased risk for coronary atherosclerosis than individuals with the wild-type genotype (OR=2.3, 95% CI=1.13-4.69). Conversely, no association between GSTM1, GSTT1, and CYP2E1gene polymorphisms and coronary atherosclerosis was detected. The results provide evidence of the role of DNA damage and repair in cardiovascular disease. © 2011 Elsevier Inc. All rights reserved.

Novos complexos de 'PD'(II): síntese, caracterização e efeito dos ligantes na citotoxicidade e interação com DNA

Pós-graduação em Química - IQ

RPA-1 from Leishmania amazonensis (LaRPA-1) structurally differs from other eukaryote RPA-1 and interacts with telomeric DNA via its N-terminal OB-fold domain

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)