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Proceedings of the National Academy of... Dec 1977A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94,...
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Topics: Base Sequence; Coliphages; DNA Polymerase I; DNA Restriction Enzymes; DNA, Viral; Deoxyribonucleotides; Methods
PubMed: 271968
DOI: 10.1073/pnas.74.12.5463 -
FASEB Journal : Official Publication of... Sep 2014For >35 yr, we have known that the accuracy of DNA replication is controlled in large part by the relative concentrations of the 4 canonical deoxyribonucleoside... (Review)
Review
For >35 yr, we have known that the accuracy of DNA replication is controlled in large part by the relative concentrations of the 4 canonical deoxyribonucleoside 5'-triphosphates (dNTPs) at the replisome. Since this field was last reviewed, ∼8 yr ago, there has been increased understanding of the mutagenic pathways as they occur in living cells. At the same time, aspects of deoxyribonucleotide metabolism have been shown to be critically involved in processes as diverse as cell cycle control, protooncogene expression, cellular defense against HIV infection, replication rate control, telomere length control, and mitochondrial function. Evidence supports a relationship between dNTP pools and microsatellite repeat instability. Relationships between dNTP synthesis and breakdown in controlling steady-state pools have become better defined. In addition, new experimental approaches have allowed definitive analysis of mutational pathways induced by dNTP pool abnormalities, both in Escherichia coli and in yeast. Finally, ribonucleoside triphosphate (rNTP) pools have been shown to be critical determinants of DNA replication fidelity. These developments are discussed in this review article.
Topics: Animals; DNA Replication; Deoxyribonucleotides; Gene Expression Regulation; Humans; Metabolic Networks and Pathways; Mutagenesis
PubMed: 24928192
DOI: 10.1096/fj.14-251249 -
The FEBS Journal May 2018The term hypoxia refers to any condition where insufficient oxygen is available and therefore encompasses a range of actual oxygen concentrations. The regions of tumours... (Review)
Review
The term hypoxia refers to any condition where insufficient oxygen is available and therefore encompasses a range of actual oxygen concentrations. The regions of tumours adjacent to necrotic areas are at almost anoxic levels and are known to be extremely therapy resistant (radiobiological hypoxia). The biological response to radiobiological hypoxia includes the rapid accumulation of replication stress and subsequent DNA damage response, including both ATR- and ATM-mediated signalling, despite the absence of detectable DNA damage. The causes and consequences of hypoxia-induced replication stress will be discussed.
Topics: Animals; Cell Cycle Proteins; Cell Hypoxia; DNA Damage; DNA Repair; DNA Replication; DNA-Binding Proteins; Deoxyribonucleotides; Humans; Neoplasms; Oxygen; Ribonucleotide Reductases; Stress, Physiological; Tumor Microenvironment
PubMed: 29288533
DOI: 10.1111/febs.14377 -
Cancer Letters Jan 2015Senescence is defined as a stable cell growth arrest. Oncogene-induced senescence (OIS) occurs when an activated oncogene is expressed in a normal cell. OIS acts as a... (Review)
Review
Senescence is defined as a stable cell growth arrest. Oncogene-induced senescence (OIS) occurs when an activated oncogene is expressed in a normal cell. OIS acts as a bona fide tumor suppressor mechanism by driving stable growth arrest of cancer progenitor cells harboring the initial oncogenic hit. OIS is often characterized by aberrant DNA replication and the associated DNA damage response. Nucleotides, in particular deoxyribonucleotide triphosphates (dNTPs), are necessary for both DNA replication and repair. Imbalanced dNTP pools play a role in a number of human diseases, including during the early stages of cancer development. This review will highlight what is currently known about the role of decreased nucleotide metabolism in OIS, how nucleotide metabolism leads to transformation and tumor progression, and how this pathway can be targeted as a cancer therapeutic by inducing senescence of cancer cells.
Topics: Biomarkers, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cellular Senescence; DNA Damage; DNA Repair; DNA Replication; Deoxyribonucleotides; Humans; Neoplasms; Neoplastic Stem Cells; Oncogenes; Signal Transduction
PubMed: 24486217
DOI: 10.1016/j.canlet.2014.01.017 -
Biochemistry May 2014This review will summarize our structural and kinetic studies of RB69 DNA polymerase (RB69pol) as well as selected variants of the wild-type enzyme that were undertaken... (Review)
Review
This review will summarize our structural and kinetic studies of RB69 DNA polymerase (RB69pol) as well as selected variants of the wild-type enzyme that were undertaken to obtain a deeper understanding of the exquisitely high fidelity of B family replicative DNA polymerases. We discuss how the structures of the various RB69pol ternary complexes can be used to rationalize the results obtained from pre-steady-state kinetic assays. Our main findings can be summarized as follows. (i) Interbase hydrogen bond interactions can increase catalytic efficiency by 5000-fold; meanwhile, base selectivity is not solely determined by the number of hydrogen bonds between the incoming dNTP and the templating base. (ii) Minor-groove hydrogen bond interactions at positions n - 1 and n - 2 of the primer strand and position n - 1 of the template strand in RB69pol ternary complexes are essential for efficient primer extension and base selectivity. (iii) Partial charge interactions among the incoming dNTP, the penultimate base pair, and the hydration shell surrounding the incoming dNTP modulate nucleotide insertion efficiency and base selectivity. (iv) Steric clashes between mismatched incoming dNTPs and templating bases with amino acid side chains in the nascent base pair binding pocket (NBP) as well as weak interactions and large gaps between the incoming dNTPs and the templating base are some of the reasons that incorrect dNTPs are incorporated so inefficiently by wild-type RB69pol. In addition, we developed a tC°-tCnitro Förster resonance energy transfer assay to monitor partitioning of the primer terminus between the polymerase and exonuclease subdomains.
Topics: Base Pairing; Catalytic Domain; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Hydrogen Bonding; Kinetics; Models, Molecular; Protein Conformation; Protein Structure, Tertiary; Substrate Specificity; Viral Proteins
PubMed: 24720884
DOI: 10.1021/bi4014215 -
The Journal of Biological Chemistry Sep 1992
Review
Topics: Base Sequence; DNA Replication; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Frameshift Mutation; Nucleic Acid Heteroduplexes
PubMed: 1526964
DOI: No ID Found -
Molecular Cancer Sep 2015Regulation of intracellular deoxynucleoside triphosphate (dNTP) pool is critical to genomic stability and cancer development. Imbalanced dNTP pools can lead to enhanced... (Review)
Review
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.
Topics: Cell Proliferation; DNA Replication; Deoxyribonucleotides; Genomic Instability; Humans; Monomeric GTP-Binding Proteins; Mutation; Neoplasms; SAM Domain and HD Domain-Containing Protein 1
PubMed: 26416562
DOI: 10.1186/s12943-015-0446-6 -
Biochemistry Feb 2011To maintain genomic stability, ribonucleotide incorporation during DNA synthesis is controlled predominantly at the DNA polymerase level. A steric clash between the... (Review)
Review
To maintain genomic stability, ribonucleotide incorporation during DNA synthesis is controlled predominantly at the DNA polymerase level. A steric clash between the 2'-hydroxyl of an incoming ribonucleotide and a bulky active site residue, known as the "steric gate", establishes an effective mechanism for most DNA polymerases to selectively insert deoxyribonucleotides. Recent kinetic, structural, and in vivo studies have illuminated novel features about ribonucleotide exclusion and the mechanistic consequences of ribonucleotide misincorporation on downstream events, such as the bypass of a ribonucleotide in a DNA template and the subsequent extension of the DNA lesion bypass product. These important findings are summarized in this review.
Topics: Animals; Carbohydrate Metabolism; Catalytic Domain; DNA; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Humans; Models, Molecular; Molecular Conformation; Substrate Specificity
PubMed: 21226515
DOI: 10.1021/bi101915z -
Pharmacology & Therapeutics Nov 2003Structural analogues of nucleosides, nucleoside analogues (NA), are used in the treatment of cancer and viral infections. Antiviral NAs inhibit replication of the viral... (Review)
Review
Structural analogues of nucleosides, nucleoside analogues (NA), are used in the treatment of cancer and viral infections. Antiviral NAs inhibit replication of the viral genome, whereas anticancer NAs inhibit cellular DNA replication and repair. NAs are inactive prodrugs that are dependent on intracellular phosphorylation to their pharmacologically active triphosphate form. The deoxyribonucleoside kinases (dNK) and ribonucleoside kinases (rNK) catalyze the first phosphorylation step, converting deoxyribonucleosides and ribonucleosides to their corresponding monophosphate form. The dNKs have been studied intensively, whereas the rNKs have not been as thoroughly investigated. This overview is focused on the substrate specificity, tissue distribution, and subcellular location of the mammalian dNKs and rNKs and their role in the activation of NAs.
Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Antiviral Agents; Deoxyribonucleotides; Humans; Molecular Sequence Data; Nucleosides; Organ Specificity; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prodrugs; Ribonucleotides; Substrate Specificity
PubMed: 14609716
DOI: 10.1016/j.pharmthera.2003.07.001 -
Cell Cycle (Georgetown, Tex.) Nov 2019Deoxyribonucleotide metabolites (dNTPs) are the substrates for DNA synthesis. It has been proposed that their availability influences the progression of the cell cycle... (Review)
Review
Deoxyribonucleotide metabolites (dNTPs) are the substrates for DNA synthesis. It has been proposed that their availability influences the progression of the cell cycle during development and pathological situations such as tumor growth. The mechanism has remained unclear for the link between cell cycle and dNTP levels beyond their role as substrates. Here, we review recent studies concerned with the dynamics of dNTP levels in early embryos and the role of DNA replication checkpoint as a sensor of dNTP levels.
Topics: Animals; Cell Cycle; Cell Division; DNA Replication; Deoxyribonucleotides; Drosophila; Metabolic Networks and Pathways; Ovum
PubMed: 31544596
DOI: 10.1080/15384101.2019.1665948