EryR mutability is influenced by replication fidelity of DNA polymerase gamma as well as by the activity of some enzymes involved in mtDNA repair and/or recombination. In an attempt to investigate about genetic conditions that might reduce the damages caused by mutations in POLG, we analyzed the effect of deletion or overexpression of TLS polymerases that, together with DNA polymerase gamma, are present in mitochondria, Pol zeta and Rev1. Genetic tests based on gene overexpression are widely used in yeast to assess a role of a gene function in a biological process. Several researches are reported where one or more proteins were overexpressed with this aim,MK-1775 955365-80-7among which a study showing that overexpression of Rev3 led to increasing UV-induced mutagenesis. In addition, gene overexpression is commonly used to rescue the phenotypic defects caused by mutations in another gene which is functionally associated. For example, in the case of MIP1, overexpression of RNR1, encoding the large subunit of the ribonucleotide reductase, lead to the discovery that increased levels of dNTP pools reduced the extended mutability caused by mutations in Mip1. This finding has had impacted also in studies on human DNA polymerase gamma pathological mutations, where, starting from analysis in yeast, it has been shown that human DNA polymerase gamma harboring the pathological mutation H932Y has a,200-fold reduced affinity to the incoming dNTPs. Here we found that increased expression of Pol zeta resulted in a significant reduction of extended mutability caused by mutations mapping in different domains of MIP1 and that overexpression of both Pol zeta and Rev1 led to a reduction in mtDNA point mutability. Petite frequency was determined as previously reported. To determine the effect of antioxidant agents on the extended mtDNA mutability, wild-type and mutant strains, were grown on liquid SCD medium supplemented with 40 mM dihydrolipoic acid or 10 mM MitoQ for two 24-hour growth cycles. Dihydrolipoic acid was supplemented from a 40 mM stock solution in ethanol, while MitoQ was supplemented from a 5 mM stock solution in DMSO. Control experiment inMK-2206 2HCl 1032349-77-1 which equal amounts of ethanol or DMSO were added to untreated cells was done in parallel. For each strain/condition at least three independent experiments were performed on three independent clones. Statistical analysis of petite frequency was performed by a two-tailed t-test. EryR mutant frequency was determined as previously reported. Briefly, 15 independent colonies, isolated on SC medium supplemented with 2% glucose, were inoculated into 2 ml of SC liquid medium supplemented with 2% ethanol and grown to saturation.