Publisher Summary This chapter discusses the purification of mammalian polymerases, DNA polymerase ɛ. Pole ɛ is highly processive in the absence of proliferating cell nuclear antigen (PCNA) and antibody raised against Pol δ does not cross-react with Pol ɛ. Pol ɛ from HeLa cells was first isolated as a repair factor required for permeabilized diploid human fibroblasts. It has also been reported that DNA repair synthesis in permeabilized yeast nuclei excision repair is catalyzed by DNA Pol ɛ. When the gene-encoding yeast Pol ɛ is disrupted, yeast cells become nonviable, indicating that Pol ɛ has a role in DNA replication. The temperature- sensitive (ts) Pole mutants of the yeast are defective in the chromosomal DNA replication at the restrictive temperature, also indicating that Pol ɛ is involved in DNA replication. DNA Pol ɛ is a processive enzyme, which can elongate several thousand nucleotides without dissociating. Indeed, Pol ɛ alone can replicate an entire DNA-primed M13 template with 1 m M MgC1 2 present. However, it requires 15 mM MgC12 for maximal velocity when assayed on poly(dA) • oligo(dT) template-primer.