Measuring deaminated nucleotide surveillance enzyme ITPA activity with an ATP-releasing nucleotide chimera

Debin Ji, Elena I. Stepchenkova, Jian Cui, Miriam R. Menezes, Youri I Pavlov, Eric T. Kool

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)11515-11524
Number of pages10
JournalNucleic acids research
Volume45
Issue number20
DOIs
StatePublished - Jan 1 2017

Fingerprint

Nucleotides
Adenosine Triphosphate
Inosine Triphosphate
Enzymes
Deamination
Adenine
Luminescence
Luciferases
Nucleosides
Mutagenesis
DNA Damage
Yeasts
DNA
Health
Research
Therapeutics

ASJC Scopus subject areas

  • Genetics

Cite this

Measuring deaminated nucleotide surveillance enzyme ITPA activity with an ATP-releasing nucleotide chimera. / Ji, Debin; Stepchenkova, Elena I.; Cui, Jian; Menezes, Miriam R.; Pavlov, Youri I; Kool, Eric T.

In: Nucleic acids research, Vol. 45, No. 20, 01.01.2017, p. 11515-11524.

Research output: Contribution to journalArticle

Ji, Debin ; Stepchenkova, Elena I. ; Cui, Jian ; Menezes, Miriam R. ; Pavlov, Youri I ; Kool, Eric T. / Measuring deaminated nucleotide surveillance enzyme ITPA activity with an ATP-releasing nucleotide chimera. In: Nucleic acids research. 2017 ; Vol. 45, No. 20. pp. 11515-11524.
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