How the cellular amount of mitochondrial DNA (mtDNA) is regulated under normal 
conditions and in the presence of genotoxic stress is less understood. We 
demonstrate that the inefficient mtDNA replication process of mutant yeast cells 
lacking the PIF1 DNA helicase is partly rescued in the absence of the DNA 
helicase RRM3. The rescue effect is likely due to the increase in the 
deoxynucleoside triphosphates (dNTPs) pool caused by the lack of RRM3. In 
contrast, the Pif1p-dependent mtDNA breakage in the presence and absence of 
genotoxic stress is not suppressed if RRM3 is lacking suggesting that this 
phenotype is likely independent of the dNTP pool. Pif1 protein (Pif1p) was found 
to stimulate the incorporation of dNTPs into newly synthesised mtDNA of 
gradient-purified mitochondria. We propose that Pif1p that acts likely as a DNA 
helicase in mitochondria affects mtDNA replication directly. Possible roles of 
Pif1p include the resolution of secondary DNA and/or DNA/RNA structures, the 
temporarily displacement of tightly bound mtDNA-binding proteins, or the 
stabilization of the mitochondrial replication complex during mtDNA replication.