Analysis of Rev1p and Pol ζ in mitochondrial mutagenesis suggests an alternative pathway of damage tolerance

Abstract

Ultraviolet light is a potent DNA damaging agent that induces bulky lesions in DNA which block the replicative polymerases. In order to ensure continued DNA replication and cell viability, specialized translesion polymerases bypass these lesions at the expense of introducing mutations in the nascent DNA strand. A recent study has shown that the N-terminal sequences of the nuclear translesion polymerases Rev1p and Pol ζ can direct GFP to the mitochondrial compartment of Saccharomyces cerevisiae. We have investigated the role of these polymerases in mitochondrial mutagenesis. Our analysis of mitochondrial DNA point mutations, microsatellite instability, and the spectra of mitochondrial mutations indicate that these translesion polymerases function in a less mutagenic pathway in the mitochondrial compartment than they do in the nucleus. Mitochondrial phenotypes resulting from the loss of Rev1p and Pol ζ suggest that although these polymerases are responsible for the majority of mitochondrial frameshift mutations, they do not greatly contribute to mitochondrial DNA point mutations. Analysis of spontaneous mitochondrial DNA point mutations suggests that Pol ζ may play a role in general mitochondrial DNA maintenance. In addition, we observe a 20-fold increase in UV-induced mitochondrial DNA point mutations in rev deficient strains. Our data provides evidence for an alternative damage tolerance pathway that is specific to the mitochondrial compartment.

Evidence That Msh1p Plays Multiple Roles in Mitochondrial Base Excision Repair

“…To determine the frequencies of mutational events responsible for Ery R with each of the msh1 alleles, the region from 1900 to 2000 of the mitochondrial 21S rRNA gene was sequenced from independently isolated Ery R colonies to determine the types of substitutions that give rise to Ery R in individual colonies. Contained within this 100-bp region is a stretch of four nucleotides whose mutation commonly confers Ery R ( Figure 1A) (Vanderstraeten et al 1998;Kalifa and Sia 2007). All sequenced Ery R clones in this study resulted from mutations in this region.…”

Mitochondrial Genome Maintenance: Roles for Nuclear Nonhomologous End-Joining Proteins inSaccharomyces cerevisiae

“…Dilutions were plated on YPG + 0.1% dextrose allowing for analysis of "petite" vs. respiring colonies. The rates of mitochondrial and nuclear point mutation were determined as previously described (Sia et al 2000;Kalifa and Sia 2007).…”

Using Genetic Reporters to Assess Stability and Mutation of the Yeast Mitochondrial Genome

“…Erythromycin binds to the 21S mitochondrial ribosomal RNA, disrupting mitochondrial protein synthesis. Specific point mutations to the mitochondrial 21S rRNA gene prevent erythromycin from binding to the gene product, conferring an erythromycin resistant (Ery R ) phenotype (Cui and Mason, 1989;Kalifa and Sia, 2007). In this way, the appearance of Ery R colonies can be used to estimate mitochondrial DNA point mutation accumulation rates using various calculation methods (Lea and Coulson, 1949;Luria and Delbruck, 1943).…”

“…In addition, in yeast, DNA damage tolerance pathways that utilize the translesion polymerase complexes encoded by Rev1p, Rev3p, and Rev7p also impact mitochondrial mutagenesis (Kalifa and Sia, 2007;Zhang et al, 2006).…”

“…To allow microsatellite instability measurement in respiring cells, a state that imposes a respiration requirement on mitochondrial DNA maintenance, we developed and characterized a version of the microsatellite reporter that is respiration competent (Kalifa and Sia, 2007;Mookerjee and Sia, 2006). This ensures that the measured microsatellite instability occurs in an otherwise functional background.…”