DNA Polymerase δ: One Polypeptide, Two Activities

Lee Pletts Goscin, John J. Byrnes

Research output: Contribution to journalArticlepeer-review

77 Scopus citations

Abstract

DNA polymerase δ from rabbit bone marrow has an associated 3′-5′-exonuclease. Previous studies demonstrated a Stokes radius of 45.5 Å by gel filtration and a sedimentation coefficient of 6.5 S by zone sedimentation. Thus, a molecular weight of 122 000 and a frictional coefficient of 1.39 were calculated [Byrnes, J. J., & Black, V. L. (1978) Biochemistry 17, 4226-4231]. Several problems obstructed further purification and definition of DNA polymerase δ. The small amount of protein obtained limited further purification as the nonspecific loss of enzyme in subsequent procedures was excessive. Furthermore, the amount of protein recovered was insufficient for conventional analysis. These difficulties have been overcome, and DNA polymerase δ has been purified to apparent homogeneity. Under conditions of nondenaturing microgel electrophoresis, DNA polymerase δ aggregates to molecular weight species of 300 000 and higher. In situ assays for DNA polymerase and exonuclease in these gels generate concordant activity profiles. Upon sodium dodecyl sulfate gel electrophoresis, δ is a single polypeptide of 122 000 apparent molecular weight. The DNA polymerase incorporates between 250 000 and 300 000 nmol of thymidine deoxyribonucleoside monophosphate (dTMP) into poly(dA)/oligo(dT) (mg of protein)-1 h-1 at 37°C; the exonuclease simultaneously hydrolyzes 13% of the newly synthesized DNA. Aphidicolin, considered to be a specific inhibitor of DNA polymerase α, inhibits both the DNA polymerase and 3′-5′-exonuclease activities of δ. DNA polymerase α from rabbit bone marrow does not share a common subunit with δ. Therefore, aphidicolin binding is not specific for α, and conclusions based upon the supposition that it is must be reconsidered.

Original languageEnglish (US)
Pages (from-to)2513-2518
Number of pages6
JournalBiochemistry
Volume21
Issue number10
DOIs
StatePublished - May 1982

ASJC Scopus subject areas

  • Biochemistry

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