Recombinant Yeast mtDNA Helicases Purification and Functional Assays

At least two DNA helicases are important for maintenance of the yeast Saccharomyces cerevisiae mitochondrial DNA (mtDNA). Pif1 protein is a 5′->3′ DNA helicase that affects the stability of mtDNA at higher temperatures but is not essential at 28�C. Pif1 is probably involved in mtDNA repair and recombination (1 ,2 ), but it also affects telomeres (3 ) and nuclear ribosomal DNA (4 ). Hmi1 protein is the second mitochondrial enzyme that has single-stranded DNA (ssDNA) stimulated ATPase and 3′->5′ DNA helicase activities. Hmi1 is essential for mtDNA maintenance in regular growth conditions (5 ). Pif1 and Hmi1 proteins are normally not abundant, thus, their overexpression is necessary for biochemical studies. In this chapter, we describe purification protocols for recombinant Hmi1 and Pif1 proteins expressed in Escherichia coli.