RESUMO
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
Genome sequence compositions and epigenetic organizations are correlated extensively across multiple length scales. Replication dynamics, in particular, is highly correlated with GC content. We combine genome-wide time of replication (ToR) data, topological domains maps and detailed functional epigenetic annotations to study the correlations between replication timing and GC content at multiple scales. We find that the decrease in genomic GC content at large scale late replicating regions can be explained by mutation bias favoring A/T nucleotide, without selection or biased gene conversion. Quantification of the free dNTP pool during the cell cycle is consistent with a mechanism involving replication-coupled mutation spectrum that favors AT nucleotides at late S-phase. We suggest that mammalian GC content composition is shaped by independent forces, globally modulating mutation bias and locally selecting on functional element. Deconvoluting these forces and analyzing them on their native scales is important for proper characterization of complex genomic correlations.
Assuntos
Replicação do DNA , Composição de Bases , Linhagem Celular Tumoral , Cromatina/genética , Evolução Molecular , Genoma Humano , Humanos , MutaçãoRESUMO
The androgen receptor is a key transcription factor contributing to the development of all stages of prostate cancer (PCa). In addition, other transcription factors have been associated with poor prognosis in PCa, amongst which c-Myc (MYC) is a well-established oncogene in many other cancers. We have previously reported that the AR promotes glycolysis and anabolic metabolism; many of these metabolic pathways are also MYC-regulated in other cancers. In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity. We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA). Treatment with MPA led to a significant reduction in cellular guanosine triphosphate (GTP) levels accompanied by nucleolar stress and p53 stabilization. In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses.
Assuntos
Nucléolo Celular/patologia , Regulação Neoplásica da Expressão Gênica/genética , IMP Desidrogenase/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Western Blotting , Imunoprecipitação da Cromatina , Humanos , Imuno-Histoquímica , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Purinas/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Receptores Androgênicos/metabolismo , Análise Serial de TecidosRESUMO
Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Mutação , Nucleotídeos/química , Fosfatos/química , Saccharomyces cerevisiae/metabolismo , Alelos , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Ciclo Celular , Análise Mutacional de DNA , Replicação do DNA , Variação Genética , Humanos , Mutagênese , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fenótipo , Plasmídeos/metabolismo , Fase S , Saccharomyces cerevisiae/genéticaRESUMO
The integrity of the genome depends on diverse pathways that regulate DNA metabolism. Defects in these pathways result in genome instability, a hallmark of cancer. Deletion of ELG1 in budding yeast, when combined with hypomorphic alleles of PCNA results in spontaneous DNA damage during S phase that elicits upregulation of ribonucleotide reductase (RNR) activity. Increased RNR activity leads to a dramatic expansion of deoxyribonucleotide (dNTP) pools in G1 that allows cells to synthesize significant fractions of the genome in the presence of hydroxyurea in the subsequent S phase. Consistent with the recognized correlation between dNTP levels and spontaneous mutation, compromising ELG1 and PCNA results in a significant increase in mutation rates. Deletion of distinct genome stability genes RAD54, RAD55, and TSA1 also results in increased dNTP levels and mutagenesis, suggesting that this is a general phenomenon. Together, our data point to a vicious circle in which mutations in gatekeeper genes give rise to genomic instability during S phase, inducing expansion of the dNTP pool, which in turn results in high levels of spontaneous mutagenesis.