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 analysis of the purified, heterodimeric native human polymerase gamma complex identified the accessory subunit as 55 kDa. We isolated the full-length cDNA encoding a 55-kDa polypeptide, expressed the cDNA in Escherichia coli and purified the 55-kDa protein to homogeneity. Recombinant Hp55 forms a high affinity, salt-stable complex with Hp140 during protein affinity chromatography. Immunoprecipitation, gel filtration, and sedimentation analyses revealed a 190-kDa complex indicative of a native heterodimer. Reconstitution of Hp140.Hp55 raises the salt optimum of Hp140, stimulates the polymerase and exonuclease activities, and increases the processivity of the enzyme by several 100-fold. Similar to Hp140, isolated Hp55 binds DNA with moderate strength and was a specificity for double-stranded primer-template DNA. However, Hp140.Hp55 has a surprisingly high affinity for DNA, and kinetic analyses indicate Hp55 enhances the affinity of Hp140 for primer termini by 2 orders of magnitude. Thus the enhanced DNA binding caused by Hp55 is the basis for the salt tolerance and high processivity characteristic of DNA polymerase gamma. Observation of native DNA polymerase gamma both as an Hp140 monomer 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-ethylmaleimide.
Lim
S E
SE
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Longley
M J
MJ
Copeland
W C
WC
D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism*; D000074002:DNA Polymerase gamma; D017931:DNA Primers; D004261:DNA Replication; D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology*; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D012965:Sodium Chloride; D004247:DNA; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D005033:Ethylmaleimide