Measuring deaminated nucleotide surveillance enzyme ITPA activity with an ATP-releasing nucleotide chimera
| Autor: | Eric T. Kool, Youri I. Pavlov, Jian Cui, Debin Ji, Elena I. Stepchenkova, Miriam R. Menezes |
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| Rok vydání: | 2017 |
| Předmět: |
Cell Extracts
0301 basic medicine DNA damage Deamination 03 medical and health sciences chemistry.chemical_compound Adenosine Triphosphate Chemical Biology and Nucleic Acid Chemistry Inosine Monophosphate Cell Line Tumor Genetics Humans Nucleotide Pyrophosphatases RNA Small Interfering Enzyme Assays Fluorescent Dyes chemistry.chemical_classification 030102 biochemistry & molecular biology biology DNA Enzyme assay 3. Good health 030104 developmental biology Enzyme Biochemistry chemistry Luminescent Measurements biology.protein RNA Interference ITPA Nucleoside Adenosine triphosphate DNA Damage HeLa Cells |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkx774 |
| Popis: | Nucleotide quality surveillance enzymes play important roles in human health, by detecting damaged molecules in the nucleotide pool and deactivating them before they are incorporated into chromosomal DNA or adversely affect metabolism. In particular, deamination of adenine moiety in (deoxy)nucleoside triphosphates, resulting in formation of (d)ITP, can be deleterious, leading to DNA damage, mutagenesis and other harmful cellular effects. The 21.5 kDa human enzyme that mitigates this damage by conversion of (d)ITP to monophosphate, ITPA, has been proposed as a possible therapeutic and diagnostic target for multiple diseases. Measuring the activity of this enzyme is useful both in basic research and in clinical applications involving this pathway, but current methods are nonselective and are not applicable to measurement of the enzyme from cells or tissues. Here, we describe the design and synthesis of an ITPA-specific chimeric dinucleotide (DIAL) that replaces the pyrophosphate leaving group of the native substrate with adenosine triphosphate, enabling sensitive detection via luciferase luminescence signaling. The probe is shown to function sensitively and selectively to quantify enzyme activity in vitro, and can be used to measure the activity of ITPA in bacterial, yeast and human cell lysates. |
| Databáze: | OpenAIRE |
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