Intradimerically tethered DNA topoisomerase II is catalytically active in DNA transport.

A covalently cross-linked dimer of yeast DNA topoisomerase II was created by fusing the enzyme with the GCN4 leucine zipper followed by two glycines and a cysteine. Upon oxidation of the chimeric protein, a disulfide bond forms between the two carboxyl termini, covalently and intradimerically cross-linking the two protomers. In addition, all nine of the cysteines naturally occurring in topoisomerase II have been changed to alanines in this construct. This cross-linked, cysteine-less topoisomerase II is catalytically active in DNA duplex passage as indicated by ATP-dependent DNA supercoil relaxation and kinetoplast DNA decatenation assays. However, these experiments do not directly distinguish between a "one-gate" and a "two-gate" mechanism for the enzyme.

Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability.

The crystal structure of the large fragment of the Thermus aquaticus DNA polymerase (Klentaq1), determined at 2.5-A resolution, demonstrates a compact two-domain architecture. The C-terminal domain is identical in fold to the equivalent region of the Klenow fragment of Escherichia coli DNA polymerase I (Klenow pol I). Although the N-terminal domain of Klentaq1 differs greatly in sequence from its counterpart in Klenow pol I, it has clearly evolved from a common ancestor. The structure of Klentaq1 reveals the strategy utilized by this protein to maintain activity at high temperatures and provides the structural basis for future improvements of the enzyme.

A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.

Bacteriophage T7 DNA polymerase efficiently incorporates a chain-terminating dideoxynucleotide into DNA, in contrast to the DNA polymerases from Escherichia coli and Thermus aquaticus. The molecular basis for this difference has been determined by constructing active site hybrids of these polymerases. A single hydroxyl group on the polypeptide chain is critical for selectivity. Replacing tyrosine-526 of T7 DNA polymerase with phenylalanine increases discrimination against the four dideoxynucleotides by > 2000-fold, while replacing the phenylalanine at the homologous position in E. coli DNA polymerase I (position 762) or T. aquaticus DNA polymerase (position 667) with tyrosine decreases discrimination against the four dideoxynucleotides 250- to 8000-fold. These mutations allow the engineering of new DNA polymerases with enhanced properties for use in DNA sequence analysis.

Human immunodeficiency virus type 1 reverse transcriptase: enhancement of activity by interaction with cellular topoisomerase I.

A number of studies have suggested that topoisomerase I (topo I) activity may be important in human immunodeficiency virus type 1 (HIV-1) replication. Specifically it has been reported that purified virus particles have topo I activity and that inhibitors of this enzyme can inhibit virus replication in vitro. We have investigated a possible association of HIV-1 gag proteins with topo I activity. We found that whereas the gag-encoded proteins by themselves do not have activity, the nucleocapsid protein p15 can interact with and enhance the activity of cellular topo I. Furthermore it could be demonstrated that topo I markedly enhanced HIV-1 reverse transcriptase activity in vitro and that this could be inhibited by the topo I-specific inhibitor camptothecin. The findings suggest that cellular topo I plays an important role in the reverse transcription of HIV-1 RNA and that the recruitment of this enzyme may be an important step in virus replication.

Fluorescent microplate-based analysis of protein-DNA interactions I:immobilized protein

A simple protein-DNA interaction analysis has been developed using a high-affinity/high-specificity zinc finger protein. In essence, purified protein samples are immobilized directly onto the surface of microplate wells, and fluorescently labeled DNA is added in solution. After incubation and washing, bound DNA is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.2 nM DNA. Since the detection of bound DNA is noninvasive and the protein-DNA interaction is not disrupted during detection, iterative readings may be taken from the same well, after successive alterations in interaction conditions, if required. In this respect, the assay may therefore be considered real time and permits appropriate interaction conditions to be determined quantitatively. The assay format is ideally suited to investigate the interactions of purified unlabeled DNA binding proteins in a high-throughput format.

Sequence-related structural DNA anomalies affect restriction enzyme activity and DNA polymerase I binding

DNA serves as a target molecule for several types of enzymes and may assume a wide variety of structural motifs depending upon the local sequence. The BssHII restriction site (GC)3 resides in a 9bp region of alternating pyrimidine and purine residues within the &phis;X174 genome. Such sequences are known to demonstrate non-canonical helical behavior under the appropriate conditions. The kinetics of BssHII cleavage was investigated in supercoiled and linear plasmid DNA, and in a 323bp DNA fragment obtained via amplification of &phis;X174. The rate of enzyme cleavage was enhanced in the supercoiled form and in the presence of 50μM cobalt hexamine. Similarly, cobalt hexamine was also found to enhance TaqI activity directly adjacent to the (GC)3 region. ^ Initial DNA polymerase I binding studies (including a gel mobility shift assay and a protection assay) indicated a notable interaction between DNA polymerase I and the BssHII site. An in-depth study revealed that equilibrium binding of DNA polymerase I to the T7 RNA polymerase promoter was comparable to that of the (GC)3 site, however the strongest interaction was observed with a cruciform containing region. Increasing the ionic strength of the solution environment, including the addition of DNA polymerase I reaction buffer significantly decreased the equilibrium dissociation constant values. ^ It is suggested that the region within or around the BssHII site experiences a conformational change generating a novel structure under the influence of supercoiled tension or 50μM cobalt hexamine. It is proposed that this transition may enhance enzyme activity and binding by providing an initial enzyme-docking site—the rate-limiting step in restriction enzyme kinetics. The high binding potential of DNA polymerase I for each of the motifs described, is hypothesized to be due to recognition of the structural DNA anomalies by the 3′–5′ exonuclease domain. ^

Mitochondrial DNA, segments I and II, its applications in population genetics and forensic sciences

The investigations of human mitochondrial DNA (mtDNA) have considerably contributed to human evolution and migration. The Middle East is considered to be an essential geographic area for human migrations out of Africa since it is located at the crossroads of Africa, and the rest of the world. United Arab Emirates (UAE) population inhabits the eastern part of Arabian Peninsula and was investigated in this study. Published data of 18 populations were included in the statistical analysis. The diversity indices showed (1) high genetic distance among African populations and (2) high genetic distance between African populations and non-African populations. Asian populations clustered together in the NJ tree between the African and European populations. MtDNA haplotypes database of the UAE population was generated. By incorporating UAE mtDNA dataset into the existing worldwide mtDNA database, UAE Forensic Laboratories will be able to analyze future mtDNA evidence in a more significant and consistent manner. ^

Role of cytokines (TNF-alpha, IL-1 beta and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide

Introduction Irinotecan (CPT-11) is an inhibitor of DNA topoisomerase I and is clinically effective against several cancers. A major toxic effect of CPT-11 is delayed diarrhea; however, the exact mechanism by which the drug induces diarrhea has not been established. Purpose Elucidate the mechanisms of induction of delayed diarrhea and determine the effects of the cytokine production inhibitor pentoxifylline (PTX) and thalidomide (TLD) in the experimental model of intestinal mucositis, induced by CPT-11. Materials and methods Intestinal mucositis was induced in male Swiss mice by intraperitoneal administration of CPT-11 (75 mg/kg) daily for 4 days. Animals received subcutaneous PTX (1.7, 5 and 15 mg/kg) or TLD (15, 30, 60 mg/kg) or 0.5 ml of saline daily for 5 and 7 days, starting 1 day before the first CPT-11 injection. The incidence of delayed diarrhea was monitored by scores and the animals were sacrificed on the 5th and 7th experimental day for histological analysis, immunohistochemistry for TNF-alpha and assay of myeloperoxidase (MPO) activity, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and KC ELISA. Results CPT-11 caused significant diarrhea, histopathological alterations (inflammatory cell infiltration, loss of crypt architecture and villus shortening) and increased intestinal tissue MPO activity, TNF-alpha, IL-1 beta and KC level and TNF-alpha immuno-staining. PTX inhibited delayed diarrhea of mice submitted to intestinal mucositis and reduced histopathological damage, intestinal MPO activity, tissue level of TNF-alpha, IL-1 beta and KC and TNF-alpha immuno-staining. TLD significantly reduced the lesions induced by CPT-11 in intestinal mucosa, decreased MPO activity, TNF-alpha tissue level and TNF-alpha immuno-staining, but did not reduce the severity of diarrhea. Conclusion These results suggest an important role of TNF-alpha, IL-1 beta and KC in the pathogenesis of intestinal mucositis induced by CPT-11.

Behavior of nuclear matrix proteins during camptothecin-induced apoptosis in HL-60 human leukemia cells.

In this study we focused our attention on the behavior of four nuclear matrix proteins during the various stages of apoptosis in the HL-60 cell line exposed to the DNA topoisomerase I inhibitor, camptothecin. We have examined the following antigens by immunocytochemical techniques: (i) the 180-kDa nucleolar isoform of DNA topoisomerase II; (ii) a 126-kDa polypeptide of nuclear bodies; (iii) a 125-kDa protein; and (iv) a 160-kDa polypeptide which are known to be components of the matrix inner network. Indirect immunofluorescence experiments were performed to follow these nuclear matrix antigens during apoptosis. Moreover, the ultrastructural localization of both 125- and 160-kDa proteins was investigated by electron microscope immunocytochemistry with gold-conjugated secondary antibodies. While the antibody to the nucleolar isoform of DNA topoisomerase II gave a fluorescent pattern that was well-maintained until the late phases of apoptosis, the other three nuclear antigens showed marked modifications in their distribution. A common feature, particularly evident for 125- and 160-kDa proteins, was their absence from cap-shaped chromatin marginations, whereas they were present in the areas of remaining decondensed chromatin. The 126-kDa polypeptide concentrated progressively in an irregular mass at the opposite side of the crescentic caps and then broke up in fine spots. The 125- and 160-kDa proteins localized in the nucleolus and precisely within certain granules which are known to appear in the nucleolar area after camptothecin administration. These results show that, in addition to the well-known chromatin changes, nuclear organization undergoes other rearrangements during the apoptotic process.

Role and Function of c-Jun Protein Complex in Cancer Cell Behavior

Transcription factors play a crucial role in the regulation of cell behavior by modulating gene expression profiles. Previous studies have described a dual role for the AP-1 family transcription factor c-Jun in the regulation of cellular fate. In various cell types weak and transient activations of c-Jun N-terminal kinase (JNK) and c-Jun appear to contribute to proliferation and survival, whereas strong and prolonged activation of JNK and c-Jun result in apoptosis. These opposite roles played by c-Jun are cell type specific and the molecular mechanisms defining these antonymous c-Jun-mediated responses remain incompletely understood. c-Jun activity in transformed cells is regulated by signalling cascades downstream of oncoproteins such as Ras and Raf. In addition, the pro-proliferative role and the survival promoting function for c-Jun has been described in various cancer models. Furthermore, c-Jun was described to be overexpressed in different cancer types. However, the molecular mechanisms by which c-Jun exerts these oncogenic functions are not all clearly established. Therefore it is of primary interest to further identify molecular mechanisms and functions for c-Jun in cancer. Regulation of gene expression is tightly dependent on accurate protein-protein interactions. Therefore, co-factors for c-Jun may define the functions for c-Jun in cancer. Identification of protein-protein interactions promoting cancer may provide novel possibilities for cancer treatment. In this study, we show that DNA topoisomerase I (TopoI) is a transcriptional co-factor for c-Jun. Moreover, c-Jun and TopoI together promote expression of epidermal growth factor receptor (EGFR) in cancer cells. We also show that the clinically used TopoI inhibitor topotecan reduces EGFR expression. Importantly, the effect of TopoI on EGFR transcription was shown to depend on c-Jun as Jun-/- cells or cells treated with JNK inhibitor SP600125 are resistant to topotecan treatment both in regulation of EGFR expression and cell proliferation. Moreover, c-Jun regulates the nucleolar localization and the function of the ribonucleic acid (RNA) helicase DDX21, a previously identified member of c-Jun protein complex. In addition, c-Jun stimulates rRNA processing by supporting DDX21 rRNA binding. Finally, this study characterizes a DDX21 dependent expression of cyclin dependent kinase (Cdk) 6, a correlation of DDX21 expression with prostate cancer progression and a substrate binding dependency of DDX21 nucleolar localization in prostate cancer cells. Taken together, the results of this study validate the c-Jun-TopoI interaction and precise the c-Jun-DDX21 interaction. Moreover, these results show the importance for protein-protein interaction in the regulation of their cellular functions in cancer cell behavior. Finally, the results presented here disclose new exciting therapeutic opportunities for cancer treatment.

Les R-loops et leurs conséquences sur l'expression génique chez Escherichia coli.

Des variations importantes du surenroulement de l’ADN peuvent être générées durant la phase d’élongation de la transcription selon le modèle du « twin supercoiled domain ». Selon ce modèle, le déplacement du complexe de transcription génère du surenroulement positif à l’avant, et du surenroulement négatif à l’arrière de l’ARN polymérase. Le rôle essentiel de la topoisomérase I chez Escherichia coli est de prévenir l’accumulation de ce surenroulement négatif générée durant la transcription. En absence de topoisomérase I, l’accumulation de ce surenroulement négatif favorise la formation de R-loops qui ont pour conséquence d’inhiber la croissance bactérienne. Les R-loops sont des hybrides ARN-ADN qui se forment entre l’ARN nouvellement synthétisé et le simple brin d’ADN complémentaire. Dans les cellules déficientes en topoisomérase I, des mutations compensatoires s’accumulent dans les gènes qui codent pour la gyrase, réduisant le niveau de surenroulement négatif du chromosome et favorisant la croissance. Une des ces mutations est une gyrase thermosensible qui s’exprime à 37 °C. La RNase HI, une enzyme qui dégrade la partie ARN d’un R-loop, peut aussi restaurer la croissance en absence de topoisomérase I lorsqu’elle est produite en très grande quantité par rapport à sa concentration physiologique. En présence de topoisomérase I, des R-loops peuvent aussi se former lorsque la RNase HI est inactive. Dans ces souches mutantes, les R-loops induisent la réponse SOS et la réplication constitutive de l’ADN (cSDR). Dans notre étude, nous montrons comment les R-loops formés en absence de topoisomérase I ou RNase HI peuvent affecter négativement la croissance des cellules. Lorsque la topoisomérase I est inactivée, l’accumulation d’hypersurenroulement négatif conduit à la formation de nombreux R-loops, ce qui déclenche la dégradation de l’ARN synthétisé. Issus de la dégradation de l’ARNm de pleine longueur, des ARNm incomplets et traductibles s’accumulent et causent l’inhibition de la synthèse protéique et de la croissance. Le processus par lequel l’ARN est dégradé n’est pas encore complètement élucidé, mais nos résultats soutiennent fortement que la RNase HI présente en concentration physiologique est responsable de ce phénotype. Chose importante, la RNase E qui est l’endoribonuclease majeure de la cellule n’est pas impliquée dans ce processus, et la dégradation de l’ARN survient avant son action. Nous montrons aussi qu’une corrélation parfaite existe entre la concentration de RNase HI, l’accumulation d’hypersurenroulement négatif et l’inhibition de la croissance bactérienne. Lorsque la RNase HI est en excès, l’accumulation de surenroulement négatif est inhibée et la croissance n’est pas affectée. L’inverse se produit Lorsque la RNase HI est en concentration physiologique. En limitant l’accumulation d’hypersurenroulement négatif, la surproduction de la RNase HI prévient alors la dégradation de l’ARN et permet la croissance. Quand la RNase HI est inactivée en présence de topoisomérase I, les R-loops réduisent le niveau d’expression de nombreux gènes, incluant des gènes de résistance aux stress comme rpoH et grpE. Cette inhibition de l’expression génique n’est pas accompagnée de la dégradation de l’ARN contrairement à ce qui se produit en absence de topoisomérase I. Dans le mutant déficient en RNase HI, la diminution de l’expression génique réduit la concentration cellulaire de différentes protéines, ce qui altère négativement le taux de croissance et affecte dramatiquement la survie des cellules exposées aux stress de hautes températures et oxydatifs. Une inactivation de RecA, le facteur essentiel qui déclenche la réponse SOS et le cSDR, ne restaure pas l’expression génique. Ceci démontre que la réponse SOS et le cSDR ne sont pas impliqués dans l’inhibition de l’expression génique en absence de RNase HI. La croissance bactérienne qui est inhibée en absence de topoisomérase I, reprend lorsque l’excès de surenroulement négatif est éliminé. En absence de RNase HI et de topoisomérase I, le surenroulement négatif est très relaxé. Il semble que la réponse cellulaire suite à la formation de R-loops, soit la relaxation du surenroulement négatif. Selon le même principe, des mutations compensatoires dans la gyrase apparaissent en absence de topoisomérase I et réduisent l’accumulation de surenroulement négatif. Ceci supporte fortement l’idée que le surenroulement négatif joue un rôle primordial dans la formation de R-loop. La régulation du surenroulement négatif de l’ADN est donc une tâche essentielle pour la cellule. Elle favorise notamment l’expression génique optimale durant la croissance et l’exposition aux stress, en limitant la formation de R-loops. La topoisomérase I et la RNase HI jouent un rôle important et complémentaire dans ce processus.

Interaction entre la RNase HI et la RNase E dans le métabolisme des R-loops et la dégradation des ARNms chez Escherichia coli.

Chez la bactérie Escherichia coli, la topoisomérase I et la gyrase représentent deux topoisomérases majeures qui participent à la régulation du surenroulement de l’ADN. Celles-ci sont codées respectivement par les gènes topA et par gyrA et gyrB. Chez les mutants topA, l’excès de surenroulement négatif qui est généré en amont de la polymérase ARN lors de la phase d’élongation de la transcription de l’ADN, entraine la formation de R-loops. Les R-loops sont des hybrides ARN-ADN qui in vivo sont formés lorsque l’ARN nouvellement transcrit forme un hybride avec le brin d’ADN matrice, le brin d’ADN complémentaire demeurant sous forme simple brin. La RNase HI est une endoribonucléase codée par le gène rnhA. Elle dégrade l’ARN de R-loops, entre autres, pour empêcher l’initiation de la réplication à des sites autres que l’origine normale, oriC. Chez les mutants rnhA, on observe une réplication indépendante de l’origine oriC. Ce type de réplication appelé cSDR, pourrait donc expliquer, du moins en partie, l’inhibition de la croissance de doubles mutants topA rnhA. A l’aide de la mutagenèse au transposon Tn5, il a été possible d’isoler des suppresseurs extragéniques qui permettaient la croissance des doubles mutants topA rnhA. Plusieurs de ces suppresseurs ont le transposon inséré dans le gène codant pour la RNase E, l’endoribonucléase principale impliquée dans la dégradation des ARNms chez E. coli. La majorité des insertions se retrouvent dans la partie C-terminale de la protéine qui est impliquée dans l’assemblage d’un complexe multiprotéique appelé l’ARN dégradosome. Les résultats obtenus démontrent que ces suppresseurs diminuent le cSDR ainsi que la réponse SOS induite constitutivement en l’absence de la RNase HI. Sachant que la RNase HI est une endoribonucléase tout comme la RNase E, une collaboration entre les deux enzymes suggère que la RNase E pourrait également jouer un rôle potentiel dans le contrôle de la formation des R-loops et bien évidemment de leur retrait au sein de la cellule. À l’opposé, il est possible que la RNase HI puisse avoir comme autre fonction la prise en charge de la maturation et de la dégradation des molécules d’ARNs.

A importância da seqüência de administração do irinotecan e 5-fluorouracil na inibição da proliferação do carcinoma de cólon humano in vitro

O câncer colorretal é um dos tumores humanos mais freqüentes e a terceira causa de morte relacionada ao câncer no mundo. Apesar de importantes progressos terapêuticos, os resultados na doença avançada ainda são muito modestos. Isto deve-se ao fato de que a droga mais utilizada nesta neoplasia, o antimetabólito 5-fluorouracil (5-FU), foi desenvolvido a mais de 40 anos produzindo taxas de respostas de somente 10-15%. Recentemente, o inibidor da topoisomerase I irinotecan (CPT-11) demonstrou, em carcinoma de cólon avançado, respostas comparáveis tanto em pacientes não tratados quanto naqueles que tiveram recaída após terapia com 5-FU. Estes resultados justificam a avaliação da combinação 5-FU/CPT-11 nesta doença. Apesar das respostas dos estudos clínicos serem promissoras, a melhor seqüência de administração destes agentes ainda não foi determinada. Neste estudo, avaliamos a combinação CPT-11/5-FU quanto ao aumento da inibição do crescimento celular quando comparada com os agentes sozinhos nas linhagens celulares de carcinoma de cólon humano SW620, HT-29 e SNU-C4. Para isto, as células foram expostas às drogas sozinhas ou a várias combinações e seqüências de dose baixa e fixa (IC20) de um dos agentes na presença de dose alta e seriada do outro. As células foram avaliadas imediatamente após os tratamentos e/ou cultivadas por mais 2 dias em meio de cultura sem drogas através de coloração com sulforodamina B. As interações entre CPT-11 e 5-FU foram avaliadas por um programa de computador que permite calcular os índices de combinação (CIs) das drogas indicando sinergismo, adição, ou antagonismo (CI < 1, = 1, ou > 1, respectivamente). As respostas celulares foram relacionadas com as atividades das enzimas timidilato sintase, topoisomerase I e carboxil esterase, que foram determinadas através de ensaio que mede os sítios de ligação e atividade catalítica da enzima, ensaio de decatenação do DNA e método espectrofotométrico, respectivamente. Estando a toxicidade no DNA envolvida no mecanismo de ação das duas drogas, também relacionamos as respostas celulares com a introdução de danos ao DNA por método fluorescente. Para melhor entendermos as interações entre as drogas, examinamos os efeitos da exposição à IC20, IC50 e/ou IC80 do CPT-11 ou 5-FU por 2 h ou 24 h em alvos celulares possivelmente relacionados com a citotoxicidade destes agentes. Estes incluem: capacidade de reparo por excisão do 10 DNA, distribuição das células nas fases do ciclo celular, integridade da membrana plasmática e formação de complexos DNA-topoisomerase I. Para isto, utilizamos método de incorporação de [3H-metil]timidina, citometria de fluxo, liberação de lactato desidrogenase (LDH) no meio de cultura e ensaio de precipitação com SDS, respectivamente. Os estudos de inibição do crescimento celular revelaram valores de IC50 do 5-FU nas linhagens SW620, HT-29 e SNU-C4 de aproximadamente 15, 8 e 2 µM, respectivamente, e do CPT-11 próximos a 2, 2 e 4 µM, respectivamente. As diferentes sensibilidades ao 5-FU nas três linhagens foram determinadas principalmente pela diferença na afinidade ao substrato. As respostas comparáveis obtidas pela exposição ao CPT-11 podem ser explicadas pelo equilíbrio entre as diferentes atividades das enzimas topoisomerase I e carboxil esterase, entre as linhagens. O programa de análise da combinação das drogas mostrou adição ou sinergismo após exposição a IC20 do CPT-11 seguido do 5-FU, nas três linhagens celulares. Por outro lado, o pré-tratamento com IC20 do 5-FU antagonizou a inibição do crescimento mediada pelo CPT-11. Nenhum dos tratamentos simultâneos determinou um aumento na inibição do crescimento nas linhagens SW620 e HT-29; mas mostraram adição ou antagonismo na linhagem SNU-C4. Observamos um significativo acréscimo na introdução de danos ao DNA nas linhagens celulares SW620 e HT-29 somente quando a IC20 do CPT-11 precedeu a IC50 do 5-FU. Já na linhagem SNU-C4, não somente este tratamento, mas também a utilização simultânea dos agentes introduziu mais dano ao DNA. Os tratamentos por 2 h ou 24 h com IC20, IC50, e/ou IC80 do 5-FU ou CPT-11 não causaram mudanças significativas na distribuição das células nas fases do ciclo celular, concentração de LDH no meio de cultura, ou formação de complexos DNA-topoisomerse I. Indicando que alterações nestes processos não estão envolvidas com os efeitos modulatórios dos pré-tratamentos na citotoxicidade das drogas. Entretanto, a incorporação de [3H-metil]timidina no DNA de células tratadas com CPT-11, aumentou em função da dose e tempo de exposição à droga. Este resultado sugere que o dano ao DNA introduzido pelo CPT-11 depende da dose e tempo de exposição podendo ser reparado por mecanismos de excisão. Somente foi observada significativa incorporação de [3H-metil]timidina em células tratadas com IC20 do 5-FU por 2 h, sugerindo que o reparo por excisão só ocorre após exposição por curto período e à doses baixas deste agente. Juntos, os resultados deste estudo mostraram que tanto os efeitos anti-proliferativos quanto a introdução de danos ao DNA pela combinação CPT-11/5-FU em dose baixa e fixa de um agente junto com dose alta do outro, nas linhagens celulares de carcinoma de cólon humano SW620, HT-29 e SNU-C4, dependem da seqüência de administração das drogas. Estes achados podem ter sido determinados pelos diferentes efeitos do tratamento com uma dose baixa dos agentes nos mecanismos de reparo por excisão do DNA.

HLA-DPB1 and DPB2 are genetic loci for systemic sclerosis: a genome-wide association study in Koreans with replication in North Americans.

OBJECTIVE: To identify systemic sclerosis (SSc) susceptibility loci via a genome-wide association study. METHODS: A genome-wide association study was performed in 137 patients with SSc and 564 controls from Korea using the Affymetrix Human SNP Array 5.0. After fine-mapping studies, the results were replicated in 1,107 SSc patients and 2,747 controls from a US Caucasian population. RESULTS: The single-nucleotide polymorphisms (SNPs) (rs3128930, rs7763822, rs7764491, rs3117230, and rs3128965) of HLA-DPB1 and DPB2 on chromosome 6 formed a distinctive peak with log P values for association with SSc susceptibility (P=8.16x10(-13)). Subtyping analysis of HLA-DPB1 showed that DPB1*1301 (P=7.61x10(-8)) and DPB1*0901 (P=2.55x10(-5)) were the subtypes most susceptible to SSc in Korean subjects. In US Caucasians, 2 pairs of SNPs, rs7763822/rs7764491 and rs3117230/rs3128965, showed strong association with SSc patients who had either circulating anti-DNA topoisomerase I (P=7.58x10(-17)/4.84x10(-16)) or anticentromere autoantibodies (P=1.12x10(-3)/3.2x10(-5)), respectively. CONCLUSION: The results of our genome-wide association study in Korean subjects indicate that the region of HLA-DPB1 and DPB2 contains the loci most susceptible to SSc in a Korean population. The confirmatory studies in US Caucasians indicate that specific SNPs of HLA-DPB1 and/or DPB2 are strongly associated with US Caucasian patients with SSc who are positive for anti-DNA topoisomerase I or anticentromere autoantibodies.

Guanine specific binding at a DNA junction formed by d[CG(5-BrU)ACG]2 with a topoisomerase poison in the presence of Co2+Ions†,‡

The structure of the duplex d[CG(5-BrU)ACG]2 bound to 9-bromophenazine-4-carboxamide has been solved through MAD phasing at 2.0 Å resolution. It shows an unexpected and previously unreported intercalation cavity stabilized by the drug and novel binding modes of Co2+ ions at certain guanine N7 sites. For the intercalation cavity the terminal cytosine is rotated to pair with the guanine of a symmetry-related duplex to create a pseudo-Holliday junction geometry, with two such cavities linked through the minor groove interactions of the N2/N3 guanine sites at an angle of 40°, creating a quadruplex-like structure. The mode of binding of the drug is shown to be disordered, with the major conformations showing the side chain bound to the N7 position of adjacent guanines. The other end of the duplex exhibits a terminal base fraying in the presence of Co2+ ions linking symmetry-related guanines, causing the helices to intertwine through the minor groove. The stabilization of the structure by the intercalating drug shows that this class of compound may bind to DNA junctions as well as duplex DNA or to strand-nicked DNA (‘hemi-intercalated'), as in the cleavable complex. This suggests a structural basis for the dual poisoning of topoisomerase I and II enzymes by this family of drugs.