ABSTRACT
Information about the intracellular concentration of dNTPs and NTPs is important for studies of the mechanisms of DNA replication and repair, but the low concentration of dNTPs and their chemical similarity to NTPs present a challenge for their measurement. Here, we describe a new rapid and sensitive method utilizing hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the simultaneous determination of dNTPs and NTPs in biological samples. The developed method showed linearity (R2 > 0.99) in wide concentration ranges and could accurately quantify dNTPs and NTPs at low pmol levels. The intra-day and inter-day precision were below 13%, and the relative recovery was between 92% and 108%. In comparison with other chromatographic methods, the current method has shorter analysis times and simpler sample pre-treatment steps, and it utilizes an ion-pair-free mobile phase that enhances mass-spectrometric detection. Using this method, we determined dNTP and NTP concentrations in actively dividing and quiescent mouse fibroblasts.
Subject(s)
Chromatography, High Pressure Liquid/methods , Deoxyribonucleotides/analysis , Ribonucleotides/analysis , Tandem Mass Spectrometry/methods , 3T3 Cells , Animals , Cell Line , Hydrophobic and Hydrophilic Interactions , MiceABSTRACT
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.
Subject(s)
Colonic Neoplasms/genetics , DNA, Neoplasm/genetics , Deoxyribonucleotides/genetics , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , Mutation/genetics , Polymorphism, Single Nucleotide/genetics , Animals , Cell Line, Tumor , DNA Replication , Genetic Predisposition to Disease/genetics , Heterozygote , Humans , Mice , Mice, Inbred C57BL , SAM Domain and HD Domain-Containing Protein 1ABSTRACT
E2F transcriptional regulators control human-cell proliferation by repressing and activating the transcription of genes required for cell-cycle progression, particularly the S phase. E2F proteins repress transcription in association with retinoblastoma pocket proteins, but less is known about how they activate transcription. Here, we show that the human G1 phase regulator HCF-1 associates with both activator (E2F1 and E2F3a) and repressor (E2F4) E2F proteins, properties that are conserved in insect cells. Human HCF-1-E2F interactions are versatile: their associations and binding to E2F-responsive promoters are cell-cycle selective, and HCF-1 displays coactivator properties when bound to the E2F1 activator and corepressor properties when bound to the E2F4 repressor. During the G1-to-S phase transition, HCF-1 recruits the mixed-lineage leukemia (MLL) and Set-1 histone H3 lysine 4 methyltransferases to E2F-responsive promoters and induces histone methylation and transcriptional activation. These results suggest that HCF-1 induces cell-cycle-specific transcriptional activation by E2F proteins to promote cell proliferation.
Subject(s)
E2F Transcription Factors/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Host Cell Factor C1/metabolism , Lysine/metabolism , Myeloid-Lymphoid Leukemia Protein/metabolism , Promoter Regions, Genetic/genetics , S Phase/genetics , Amino Acid Sequence , Conserved Sequence , E2F Transcription Factors/chemistry , Evolution, Molecular , G1 Phase , HeLa Cells , Histone Deacetylases/metabolism , Humans , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Repressor Proteins/metabolism , Retinoblastoma Protein/metabolism , Retinoblastoma-Like Protein p130/metabolism , Sin3 Histone Deacetylase and Corepressor Complex , Two-Hybrid System TechniquesABSTRACT
Ribonucleotide reductase is essential for supplying a balanced pool of the four deoxyribonucleotides required for DNA synthesis and repair. The active enzyme consists of two non-identical subunits called proteins R1 and R2. There are multiple levels of regulation of ribonucleotide reductase activity, which is highest during the S and G(2) phases of an unperturbed cell cycle in mammalian cells. Previous reports in the literature have indicated that the S phase-specific transcription of the mammalian R2 gene is regulated by a transcriptional block, is dependent on the transcription factor E2F1, or is simply regulated by proteins that bind to promoter CCAAT boxes plus the TATA box. Here, we demonstrate that the S phase-specific transcription of the mouse R2 gene is dependent on an upstream promoter activating region (located at nucleotides (nt) -672 to -527 from the transcription start site) and one proximal promoter repressive element (located at nt -112 to -107) that binds E2F4. Binding to the E2F site is modulated by binding of nuclear factor-Y to an adjacent CCAAT element (nt -79 to -75). The upstream activating region is crucial for overall R2 promoter activity. Mutation of the E2F-binding site leads to premature promoter activation in G(1) and increases overall promoter activity but only when the upstream activating region is present and intact. Therefore, E2F-dependent repression is essential for cell cycle-specific R2 transcription.
Subject(s)
Cell Cycle Proteins , Promoter Regions, Genetic , Ribonucleotide Reductases/biosynthesis , S Phase , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Cell Line , Chromatin/metabolism , Cloning, Molecular , DNA-Binding Proteins/chemistry , Deoxyribonuclease I/metabolism , E2F Transcription Factors , E2F1 Transcription Factor , E2F4 Transcription Factor , G1 Phase , G2 Phase , Gene Deletion , Gene Expression Regulation , Guinea Pigs , Humans , Luciferases/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mutation , Polymerase Chain Reaction , Precipitin Tests , Ribonucleotide Reductases/genetics , Time Factors , Transcription Factors/chemistry , Transcription, Genetic , TransfectionABSTRACT
Ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, and catalyzes the rate-limiting step in DNA precursor synthesis: the reduction of ribonucleotides to deoxyribonucleotides. A strictly balanced supply of deoxyribonucleotides is essential for both accurate DNA replication and repair. Therefore, ribonucleotide reductase activity is under exquisite control both transcriptionally and posttranscriptionally. In proliferating mammalian cells, enzyme activity is regulated by control of R2 protein stability. This control, which responds to DNA damage, is effective until cells pass into mitosis. We demonstrate that the mitotic degradation and hence the overall periodicity of R2 protein levels depends on a KEN box sequence, recognized by the Cdh1-anaphase-promoting complex. The mouse R2 protein specifically binds Cdh1 and is polyubiquitinated in an in vitro ubiquitin assay system. Mutating the KEN signal stabilizes the R2 protein during mitosisG(1) in R2 protein-overexpressing cells. The degradation process, which blocks deoxyribonucleotide production during G(1), may be an important mechanism protecting the cell against unscheduled DNA synthesis. The newly discovered p53-induced p53R2 protein that lacks a KEN box may supply deoxyribonucleotides for DNA repair during G(0)G(1).
