RESUMO
Stimulated by the growing interest in the role of dNTP pools in physiological and malignant processes, we established dNTPpoolDB, the database that offers access to quantitative data on dNTP pools from a wide range of species, experimental and developmental conditions (https://dntppool.org/). The database includes measured absolute or relative cellular levels of the four canonical building blocks of DNA and of exotic dNTPs, as well. In addition to the measured quantity, dNTPpoolDB contains ample information on sample source, dNTP quantitation methods and experimental conditions including any treatments and genetic manipulations. Functions such as the advanced search offering multiple choices from custom-built controlled vocabularies in 15 categories in parallel, the pairwise comparison of any chosen pools, and control-treatment correlations provide users with the possibility to quickly recognize and graphically analyse changes in the dNTP pools in function of a chosen parameter. Unbalanced dNTP pools, as well as the balanced accumulation or depletion of all four dNTPs result in genomic instability. Accordingly, key roles of dNTP pool homeostasis have been demonstrated in cancer progression, development, ageing and viral infections among others. dNTPpoolDB is designated to promote research in these fields and fills a longstanding gap in genome metabolism research.
Assuntos
Bases de Dados Genéticas , Desoxirribonucleotídeos/classificação , Instabilidade Genômica/genética , Neoplasias/genética , Replicação do DNA/genética , Curadoria de Dados , Desoxirribonucleotídeos/genética , Humanos , Neoplasias/classificação , Neoplasias/patologiaRESUMO
Deoxyribonucleotide biosynthesis from ribonucleotides supports the growth of active cancer cells by producing building blocks for DNA. Although ribonucleotide reductase (RNR) is known to catalyze the rate-limiting step of de novo deoxyribonucleotide triphosphate (dNTP) synthesis, the biological function of the RNR large subunit (RRM1) in small-cell lung carcinoma (SCLC) remains unclear. In this study, we established siRNA-transfected SCLC cell lines to investigate the anticancer effect of silencing RRM1 gene expression. We found that RRM1 is required for the full growth of SCLC cells both in vitro and in vivo. In particular, the deletion of RRM1 induced a DNA damage response in SCLC cells and decreased the number of cells with S phase cell cycle arrest. We also elucidated the overall changes in the metabolic profile of SCLC cells caused by RRM1 deletion. Together, our findings reveal a relationship between the deoxyribonucleotide biosynthesis axis and key metabolic changes in SCLC, which may indicate a possible link between tumor growth and the regulation of deoxyribonucleotide metabolism in SCLC.
Assuntos
Proliferação de Células , Desoxirribonucleotídeos/biossíntese , Neoplasias Pulmonares/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Animais , Linhagem Celular Tumoral , Dano ao DNA , Desoxirribonucleotídeos/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleosídeo Difosfato Redutase/metabolismo , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologiaRESUMO
Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Desoxirribonucleotídeos/isolamento & purificação , Oligonucleotídeos/genética , Animais , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/química , Desoxirribonucleotídeos/genética , Camundongos , Inibidores da Síntese de Ácido Nucleico/química , Oligonucleotídeos/síntese química , Ribonuclease H/genéticaRESUMO
Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi-Goutières syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c-Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break-point junctions is a notable feature in SAMHD1 deficiency during activation-induced cytidine deaminase-mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double-stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1-mediated dNTP balance regulates dNTP-sensitive DNA end-processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway.
Assuntos
Reparo do DNA , Desoxirribonucleotídeos/metabolismo , Switching de Imunoglobulina , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Linhagem Celular , Desoxirribonucleotídeos/genética , Humanos , Proteína 1 com Domínio SAM e Domínio HD/genéticaRESUMO
Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain- and HD domain-containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase-mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for â¼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages.
Assuntos
Infecções por HIV/metabolismo , HIV/metabolismo , Macrófagos/virologia , Transcrição Reversa , Ribonucleotídeos/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Células Cultivadas , Desoxirribonucleotídeos/genética , Desoxirribonucleotídeos/metabolismo , HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , HIV-2/genética , HIV-2/metabolismo , Humanos , Células Jurkat , Macrófagos/metabolismo , Mutagênese , Ribonucleotídeos/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismoRESUMO
Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate ß subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a MnII2 cluster by free superoxide to yield a metal-based MnIIIMnIV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive ß subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.
Assuntos
Domínio Catalítico , Desoxirribonucleotídeos/química , Conformação Proteica , Ribonucleotídeo Redutases/química , Actinobacillus/enzimologia , Actinobacillus/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , Desoxirribonucleotídeos/biossíntese , Desoxirribonucleotídeos/genética , Flavobacterium/enzimologia , Flavobacterium/genética , Modelos Moleculares , Filogenia , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/genética , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Difração de Raios XRESUMO
Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex.
Assuntos
Algoritmos , DNA Viral/química , Vírus da Leucemia Murina/enzimologia , Modelos Químicos , RNA Viral/química , DNA Polimerase Dirigida por RNA/química , DNA Viral/genética , DNA Viral/metabolismo , Desoxirribonucleotídeos/genética , Desoxirribonucleotídeos/metabolismo , Cinética , Vírus da Leucemia Murina/genética , Pinças Ópticas , Polimerização , RNA Viral/genética , RNA Viral/metabolismo , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Moldes Genéticos , TermodinâmicaRESUMO
Even small variations in dNTP concentrations decrease DNA replication fidelity, and this observation prompted us to analyze genomic cancer data for mutations in enzymes involved in dNTP metabolism. We found that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), a deoxyribonucleoside triphosphate triphosphohydrolase that decreases dNTP pools, is frequently mutated in colon cancers, that these mutations negatively affect SAMHD1 activity, and that several SAMHD1 mutations are found in tumors with defective mismatch repair. We show that minor changes in dNTP pools in combination with inactivated mismatch repair dramatically increase mutation rates. Determination of dNTP pools in mouse embryos revealed that inactivation of one SAMHD1 allele is sufficient to elevate dNTP pools. These observations suggest that heterozygous cancer-associated SAMHD1 mutations increase mutation rates in cancer cells.
Assuntos
Neoplasias do Colo/genética , DNA de Neoplasias/genética , Desoxirribonucleotídeos/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Animais , Linhagem Celular Tumoral , Replicação do DNA , Predisposição Genética para Doença/genética , Heterozigoto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína 1 com Domínio SAM e Domínio HDRESUMO
Regulation of intracellular deoxynucleoside triphosphate (dNTP) pool is critical to genomic stability and cancer development. Imbalanced dNTP pools can lead to enhanced mutagenesis and cell proliferation resulting in cancer development. Therapeutic agents that target dNTP synthesis and metabolism are commonly used in treatment of several types of cancer. Despite several studies, the molecular mechanisms that regulate the intracellular dNTP levels and maintain their homeostasis are not completely understood. The discovery of SAMHD1 as the first mammalian dNTP triphosphohydrolase provided new insight into the mechanisms of dNTP regulation. SAMHD1 maintains the homeostatic dNTP levels that regulate DNA replication and damage repair. Recent progress indicates that gene mutations and epigenetic mechanisms lead to downregulation of SAMHD1 activity or expression in multiple cancers. Impaired SAMHD1 function can cause increased dNTP pool resulting in genomic instability and cell-cycle progression, thereby facilitating cancer cell proliferation. This review summarizes the latest advances in understanding the importance of dNTP metabolism in cancer development and the novel function of SAMHD1 in regulating this process.
Assuntos
Desoxirribonucleotídeos/metabolismo , Instabilidade Genômica , Proteínas Monoméricas de Ligação ao GTP/genética , Neoplasias/genética , Proliferação de Células , Replicação do DNA/genética , Desoxirribonucleotídeos/genética , Humanos , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Proteína 1 com Domínio SAM e Domínio HDRESUMO
Macrophages are a major target cell for HIV-1, and their infection contributes to HIV pathogenesis. We have previously shown that the cyclin-dependent kinase inhibitor p21 inhibits the replication of HIV-1 and other primate lentiviruses in human monocyte-derived macrophages by impairing reverse transcription of the viral genome. In the attempt to understand the p21-mediated restriction mechanisms, we found that p21 impairs HIV-1 and simian immunodeficiency virus (SIV)mac reverse transcription in macrophages by reducing the intracellular deoxyribonucleotide (dNTP) pool to levels below those required for viral cDNA synthesis by a SAM domain and HD domain-containing protein 1 (SAMHD1)-independent pathway. We found that p21 blocks dNTP biosynthesis by down-regulating the expression of the RNR2 subunit of ribonucleotide reductase, an enzyme essential for the reduction of ribonucleotides to dNTP. p21 inhibits RNR2 transcription by repressing E2F1 transcription factor, its transcriptional activator. Our findings unravel a cellular pathway that restricts HIV-1 and other primate lentiviruses by affecting dNTP synthesis, thereby pointing to new potential cellular targets for anti-HIV therapeutic strategies.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Desoxirribonucleotídeos/biossíntese , Regulação Enzimológica da Expressão Gênica , Infecções por HIV/metabolismo , HIV-1/fisiologia , Macrófagos/metabolismo , Ribonucleotídeo Redutases/biossíntese , Replicação Viral/fisiologia , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , DNA Complementar/biossíntese , DNA Complementar/genética , DNA Viral/biossíntese , DNA Viral/genética , Desoxirribonucleotídeos/genética , Regulação para Baixo/genética , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Infecções por HIV/terapia , Infecções por HIV/virologia , Macrófagos/virologia , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ribonucleotídeo Redutases/genética , Proteína 1 com Domínio SAM e Domínio HD , Vírus da Imunodeficiência Símia/fisiologia , Transcrição Gênica/genéticaRESUMO
In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS.
Assuntos
Senescência Celular/genética , Dano ao DNA/genética , Desoxirribonucleotídeos/metabolismo , Oncogenes/fisiologia , Proliferação de Células , Células Cultivadas , Senescência Celular/fisiologia , Replicação do DNA/genética , Desoxirribonucleotídeos/genética , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Ribonucleotídeo Redutases/biossíntese , Ribonucleotídeo Redutases/fisiologia , Timidilato Sintase/biossíntese , Timidilato Sintase/fisiologiaRESUMO
We simulated in our laboratory a prebiotic environment where dry and wet periods were cycled. Under anhydrous conditions, lipid molecules present in the medium could form fluid lamellar matrices and work as organizing agents for the condensation of nucleic acid monomers into polymers. We exposed a mixture of 2'-deoxyribonucleoside 5'-monophosphates and a ssDNA oligomer template to this dry environment at 90 °C under a continuous gentle stream of CO(2) and we followed it with rehydration periods. After five dry/wet cycles we were able to detect the presence of a product that was complementary to the template. The reaction had a 0.5% yield with respect to the template, as measured by staining with the Pico Green(®) fluorescent probe. Absent initial template, the product of the reaction remained below the detection limit. In order to characterize the fidelity of replication, the synthesized strand was ligated to adapters, amplified by PCR, and sequenced. The alignment of the sequenced DNA to the expected complementary sequence revealed that the misincorporation rate was 9.9%. We present these results as a proof of concept for the possibility of having non-enzymatic transfer of sequence information in a prebiotically plausible environment.
Assuntos
Replicação do DNA , Adenosina/análogos & derivados , Adenosina/química , Sequência de Bases , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Desoxirribonucleotídeos/biossíntese , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/genética , Glicerofosfolipídeos/química , Modelos Moleculares , Conformação de Ácido Nucleico , Polimerização , TemperaturaRESUMO
Elevated circulating glucose resulting from complications of obesity and metabolic disease can result in the accumulation of advanced glycation end products (AGEs) of proteins, lipids, and DNA. The formation of DNA-AGEs assumes particular importance as these adducts may contribute to genetic instability and elevated cancer risk associated with metabolic disease. The principal DNA-AGE, N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG), is formed as a mixture of R and S isomers at both the polymer and monomer levels. In order to examine the miscoding potential of this adduct, oligonucleotides substituted with (R)- and (S)-CEdG and the corresponding triphosphates (R)- and (S)-CEdGTP were synthesized, and base-pairing preferences for each stereoisomer were examined using steady-state kinetic approaches. Purine dNTPs were preferentially incorporated opposite template CEdG when either the Klenow (Kf(-)) or Thermus aquaticus (Taq) polymerases were used. The Kf(-) polymerase preferentially incorporated dGTP, whereas Taq demonstrated a bias for dATP. Kf(-) incorporated purines opposite the R isomer with greater efficiency, but Taq favored the S isomer. Incorporation of (R)- and (S)-CEdGTP only occurred opposite dC and was catalyzed by Kf(-) with equal efficiencies. Primer extension from a 3'-terminal CEdG was observed only for the R isomer. These data suggest CEdG is the likely adduct responsible for the observed pattern of G transversions induced by exposure to elevated glucose or its alpha-oxoaldehyde decomposition product methylglyoxal. The results imply that CEdG within template DNA and the corresponding triphosphate possess different syn/anti conformations during replication which influence base-pairing preferences. The implications for CEdG-induced mutagenesis in vivo are discussed.
Assuntos
Pareamento Incorreto de Bases/genética , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/genética , Guanosina/análogos & derivados , Mutagênicos/síntese química , Catálise , Adutos de DNA/síntese química , Adutos de DNA/genética , Adutos de DNA/metabolismo , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxicitosina/genética , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/genética , Desoxirribonucleotídeos/metabolismo , Glicosilação , Guanosina/síntese química , Guanosina/genética , Guanosina/metabolismo , Humanos , Testes de Mutagenicidade , Mutagênicos/metabolismo , Estereoisomerismo , Moldes GenéticosRESUMO
DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Reparo do DNA , DNA/metabolismo , Desoxirribonucleotídeos/genética , Proteínas/metabolismo , Recombinação Genética , Animais , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteína BRCA2/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromossomos/metabolismo , Cricetinae , DNA/química , DNA/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Decitabina , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Formaldeído/farmacologia , Histonas/metabolismo , Humanos , Peso Molecular , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/química , Rad51 Recombinase/metabolismoRESUMO
Our recent observation that hepatitis B virus (HBV) DNA polymerase (P) might initiate minus-strand DNA synthesis without primer [Kim et al., (2004) Virology 322, 22-30], raised a possibility that HBV P protein may have the potential to function as an RNA polymerase. Thus, we mutated Phe 436, a bulky amino acid with aromatic side chain, at the putative dNTP-binding cleft in reverse transcriptase (RT) domain of P protein to smaller amino acids (Gly or Val), and examined RNA polymerase activity. HBV core particles containing RT dNTP-binding cleft mutant P protein were able to incorporate (32)P-ribonucleotides, but not HBV core particles containing wild type (wt), priming-deficient mutant, or RT-deficient mutant P proteins. Since all the experiments were conducted with core particles isolated from transfected cells, our results indicate that the HBV RT mutant core particles containing RT dNTP-binding cleft mutant P protein could incorporate both deoxyribonucleotides and ribonucleotides in replicating systems.
Assuntos
Desoxirribonucleotídeos/metabolismo , Vírus da Hepatite B/enzimologia , Mutação , DNA Polimerase Dirigida por RNA/genética , Ribonucleotídeos/metabolismo , Vírion/metabolismo , Linhagem Celular Tumoral , Desoxirribonucleotídeos/genética , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Vírus da Hepatite B/genética , Humanos , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Ribonucleotídeos/genéticaRESUMO
The SWI/SNF chromatin remodeling complex plays a critical role in the coordination of gene expression with physiological stimuli. The synthetic enzymes ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase are coordinately regulated to ensure appropriate deoxyribonucleotide triphosphate levels. Particularly, these enzymes are actively repressed as cells exit the cell cycle through the action of E2F transcription factors and the retinoblastoma tumor suppressor/p107/p130 family of pocket proteins. This process is found to be highly dependent on SWI/SNF activity as cells deficient in BRG-1 and Brm subunits fail to repress these genes with activation of pocket proteins, and this deficit in repression can be complemented, via the ectopic expression of BRG-1. The failure to repress transcription does not involve a blockade in the association of E2F or pocket proteins p107 and p130 with promoter elements. Rather, the deficit in repression is due to a failure to mediate histone deacetylation of ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase promoters in the absence of SWI/SNF activity. The basis for this is found to be a failure to recruit mSin3B and histone deacetylase proteins to promoters. Thus, the coordinate repression of deoxyribonucleotide triphosphate metabolic enzymes is dependent on the action of SWI/SNF in facilitating the assembly of repressor complexes at the promoter.
Assuntos
Ciclo Celular/fisiologia , Montagem e Desmontagem da Cromatina/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Desoxirribonucleotídeos/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , DNA Helicases/deficiência , DNA Helicases/metabolismo , Desoxirribonucleotídeos/genética , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Elementos de Resposta/fisiologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/genética , Proteína p130 Retinoblastoma-Like/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Intracellular concentrations of the four deoxyribonucleoside triphosphates (dNTPs) are closely regulated, and imbalances in the four dNTP pools have genotoxic consequences. Replication errors leading to mutations can occur, for example, if one dNTP in excess drives formation of a non-Watson-Crick base pair or if it forces replicative DNA chain elongation past a mismatch before DNA polymerase can correct the error by 3' exonuclease proofreading. This review focuses on developments since 1994, when the field was last reviewed comprehensively. Emphasis is placed on the following topics: 1) novel aspects of dNTP pool regulation, 2) dNTP pool asymmetries as mutagenic determinants, 3) dNTP metabolism and hypermutagenesis of retroviral genomes, 4) dNTP metabolism and mutagenesis in the mitochondrial genome, 5) chemical modification of nucleotides as a premutagenic event, 6) relationships between dNTP metabolism, genome stability, aging, and cancer.
Assuntos
Replicação do DNA , Desoxirribonucleotídeos/metabolismo , Genoma , Animais , Dano ao DNA , DNA Mitocondrial/genética , Desoxirribonucleotídeos/genética , Regulação da Expressão Gênica , Genoma Viral , Humanos , Mutagênese , Oncogenes , Retroviridae/genéticaRESUMO
PURPOSE: Mutations in the fibroblast growth factor receptor 3 (FGFR3) occur in 50% of primary bladder tumors. An FGFR3 mutation is associated with good prognosis, illustrated by significantly lower percentage of patients with progression and disease-specific mortality. FGFR3 mutations are especially prevalent in low grade/stage tumors, with pTa tumors harboring mutations in 85% of the cases. These tumors recur in 70% of patients. Efficient FGFR3 mutation detection for prognostic purposes and for detection of recurrences in urine is an important clinical issue. In this paper, we describe a simple assay for the simultaneous detection of nine different FGFR3 mutations. EXPERIMENTAL DESIGN: The assay consists of one multiplex PCR, followed by extension of primers for each mutation with a labeled dideoxynucleotide. The extended primers are separated by capillary electrophoresis, and the identity of the incorporated nucleotide indicates the presence or absence of a mutation. RESULTS: The assay was found to be more sensitive than single-strand conformation polymorphism analysis. Mutations could still be detected with an input of only 1 ng of genomic DNA and in a 20-fold excess of wild-type DNA. Moreover, in urine samples from patients with a mutant tumor, the sensitivity of mutation detection was 62%. CONCLUSIONS: We have developed a fast, easy to use assay for the simultaneous detection of FGFR3 mutations, which can be of assistance in clinical decision-making and as an alternative for the follow-up of patients by invasive cystoscopy for the detection of recurrences in urine.
Assuntos
Mutação , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/urina , DNA/metabolismo , Análise Mutacional de DNA , Primers do DNA/genética , Desoxirribonucleotídeos/genética , Eletroforese Capilar , Humanos , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Prognóstico , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
To broaden the applicability of chemically modified DNAs in nano- and biotechnology, material science, sensor development, and molecular recognition, strategies are required for introducing a large variety of different modifications into the same nucleic acid sequence at once. Here, we investigate the scope and limits for obtaining functionalized dsDNA by primer extension and PCR, using a broad variety of chemically modified deoxynucleotide triphosphates (dNTPs), DNA polymerases, and templates. All natural nucleobases in each strand were substituted with up to four different base-modified analogues. We studied the sequence dependence of enzymatic amplification to yield high-density functionalized DNA (fDNA) from modified dNTPs, and of fDNA templates, and found that GC-rich sequences are amplified with decreased efficiency as compared to AT-rich ones. There is also a strong dependence on the polymerase used. While family A polymerases generally performed poorly on "demanding" templates containing consecutive stretches of a particular base, family B polymerases were better suited for this purpose, in particular Pwo and Vent (exo-) DNA polymerase. A systematic analysis of fDNAs modified at increasing densities by CD spectroscopy revealed that single modified bases do not alter the overall B-type DNA structure, regardless of their chemical nature. A density of three modified bases induces conformational changes in the double helix, reflected by an inversion of the CD spectra. Our study provides a basis for establishing a generally applicable toolbox of enzymes, templates, and monomers for generating high-density functionalized DNAs for a broad range of applications.
Assuntos
Biotecnologia , DNA Polimerase Dirigida por DNA/metabolismo , DNA , Sequência de Bases , Dicroísmo Circular/métodos , DNA/química , DNA/genética , DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/genética , Desoxirribonucleotídeos/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Nanotecnologia , Reação em Cadeia da Polimerase/métodos , Polifosfatos/química , Moldes GenéticosRESUMO
To evaluate the in vivo mutagenicities of damaged DNA precursors (deoxyribonucleoside 5'-triphosphates) produced by exposure to nitric oxide (NO) and ionizing radiation, five damaged deoxyribonucleotides (deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, dUTP, and 8-hydroxy-dATP) were introduced into competent Escherichia coli cells. Their mutagenic potentials were assayed using the chromosomal rpoB gene as a mutagenesis target. In contrast to 8-hydroxy-dGTP and 2-hydroxy-dATP, which were examined in an earlier study, none of these damaged deoxyribonucleotides significantly increased the rpoB mutant frequency. These results suggest that these five damaged deoxyribonucleotides are weakly mutagenic in vivo if at all. Thus their contributions to mutations induced by NO and ionizing radiation may be small.