Pivotal Role of Inosine Triphosphate Pyrophosphatase in Maintaining Genome Stability and the Prevention of Apoptosis in Human Cells

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

A Step-by-Step Protocol for COMPASS, a Synthetic Biology Tool for Combinatorial Gene Assembly

For industry-scale production of high-value chemicals in microbial cell factories, the elimination of metabolic flux imbalances is a critical aspect. However, a priori knowledge about the genetic design of optimal production pathways is typically not available. COMPASS, COMbinatorial Pathway ASSembly, is a rapid cloning method for the balanced expression of multiple genes in biochemical pathways. The method generates thousands of individual DNA constructs in modular, parallel, and high-throughput manner. COMPASS employs inducible artificial transcription factors derived from plant (Arabidopsis thaliana) regulators to control the expression of pathway genes in yeast (Saccharomyces cerevisiae). It utilizes homologous recombination for parts assembly and employs a positive selection scheme to identify correctly assembled pathway variants after both in vivo and in vitro recombination. Finally, COMPASS is equipped with a CRISPR/Cas9 genome modification system allowing for the one-step multilocus integration of genes. Although COMPASS was initially developed for pathway engineering, it can equally be employed for balancing gene expression in other synthetic biology projects