The mitochondrial p55 accessory subunit of human DNA polymerase gamma enhances DNA binding, promotes processive DNA synthesis, and confers N-ethylmaleimide resistance

Human DNA polymerase gamma is composed of a 140-kDa catalytic subunit and a smaller accessory protein variously reported to be 43-54 kDa. Immunoblot a nalysis of the purified, heterodimeric native human polymerase gamma comple x identified the accessory subunit as 55 kDa, We isolated the full-length c DNA encoding a 55-kDa polypeptide, expressed the cDNA in Escherichia coli a nd purified the 55-kDa protein to homogeneity, Recombinant Hp55 forms a hig h affinity, salt-stable complex with Hp140 during protein affinity chromato graphy. Immunoprecipitation, gel filtration, and sedimentation analyses rev ealed a 190-kDa complex indicative of a native heterodimer, Reconstitution of Hp140 Hp55 raises the salt optimum of Hp140, stimulates the polymerase a nd exonuclease activities, and increases the processivity of the enzyme by several 100-fold. Similar to Hp140, isolated Hp55 binds DNA with moderate s trength and was a specificity for double-stranded primer-template DNA. Howe ver, Hp140 Hp55 has a surprisingly high affinity for DNA, and kinetic analy ses indicate Hp55 enhances the affinity of Hp140 for primer termini by 2 or ders of magnitude. Thus the enhanced DNA binding caused by Hp55 is the basi s for the salt tolerance and high processivity characteristic of DNA polyme rase gamma, Observation of native DNA polymerase gamma both as an Hp140 mon omer and as a heterodimer with Hp55 supports the notion that the two forms act in mitochondrial DNA repair and replication. Additionally, association of Hp55 with Hp140 protects the polymerase from inhibition by N-ethylmaleim ide.