Abstract
Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer.
Highlights
The human genome is constantly attacked by exogenous or endogenous DNA damaging agents
An understanding of the role of Inosine Triphosphate Pyrophosphatase (ITPA) in human cells is important because several alleles representing polymorphism in the ITPA gene are associated with the onset of thiopurine therapyrelated diseases
We probed the function of ITPA by determining whether the enzyme protected HeLa cells against the harmful effects of the model purine analog, HAP
Summary
The human genome is constantly attacked by exogenous or endogenous DNA damaging agents. A prominent cause of endogenous DNA damage decreasing the fidelity of DNA replication is contamination of the nucleotide precursor pool with non-canonical nucleotides [3,4]. Taking into consideration the harmful effects of base analog incorporation, it is not surprising that cells have developed elaborate enzymatic systems that protect from base analog-induced DNA damage [12,13]. The second level involves detection of improper bases after incorporation and their direct removal from DNA The former is achieved by a class of enzymes called nucleoside triphosphatases (NTPases) [3]. One such NTPase is evolutionary conserved Inosine Triphosphate Pyrophosphatase (ITPA) [14]
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