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The pool of free intracellular Mg varies among tissues with the highest concentration measured in muscle tissue and the lowest measured in immune cells and in the brain. Here we investigate the impact of free Mg on the fidelity of human DNA ligase I (LIG1). LIG1 is the major DNA ligase and is required to complete DNA replication, recombination and repair pathways. Biallelic hypomorphic variants of LIG1 cause immunodeficiency-96 (IMD96). We employed steady-state kinetics to compare fidelity of LIG1 towards a damaged nucleobase at the 3´-OH side of a nicked DNA substrate. The fidelity for discrimination between a damaged and undamaged nick increases by a factor of 21-fold when the free Mg concentration is decreased from 1.0 to 0.2 mM. This has important implications for neurodegenerative and immune diseases, because the brain and the immune system are reported to have free Mg concentration in the range from 0.2 - 0.4 mM. We examined a recently characterized minor variant of LIG1, K845N, which has a protective effect in Huntington's disease, and found that the fidelity of K845N LIG1 is also enhanced as free Mg decreases. This increase in fidelity is mainly due to the increased release of the AMP-DNA intermediate from a pro-mutagenic DNA substrate. A model is proposed whereby the fidelity of DNA transactions is sensitive to the availability of the Mg cofactor for DNA ligation and therefore ligation fidelity may vary between tissues.