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J Biol Chem, Vol. 274, Issue 53, 38197-38203, December 31, 1999
From the Laboratory of Molecular Genetics, NIEHS, National
Institutes of Health,
Research Triangle Park, North Carolina 27709
Human DNA polymerase The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF142992.
The Mitochondrial p55 Accessory Subunit of Human DNA Polymerase
Enhances DNA Binding, Promotes Processive DNA Synthesis, and
Confers N-Ethylmaleimide Resistance*
,, and
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 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 . Observation of native DNA polymerase 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.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
These authors contributed equally to this work.
§
To whom correspondence should be addressed: Laboratory of Molecular
Genetics, NIEHS, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-4792; Fax: 919-541-7613; E-mail: copelan1@niehs.nih.gov.
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