Structural study of immunoaffinity-purified DNA polymerase alpha-DNA primase complex from calf thymus.
The DNA polymerase alpha-DNA primase complex was purified over 17,000-fold to near homogeneity from calf thymus using an immunoaffinity column. Sodium dodecyl sulfate gel electrophoresis revealed three polypeptides with molecular weights of 140, 50 and 47 kDa, in a ratio of 1:2:0.25. The complex showed a sedimentation coefficient of 9.7 S, a Stokes radius of 56 A and a native molecular weight of 250-260 kDa. Taken together, the data suggest that the calf thymus dNA polymerase alpha-DNA primase complex is essentially a heterotrimer of large (140 kDa) and small (50 kDa) subunits in a ratio of 1:2, with a globular conformation. Electron-microscopic studies of the complex revealed a spherical particle of 120 A in diameter, in agreement with the physiochemical results. The binding of the complex to DNA was also demonstrated.
Tamai
K
K
Laboratory of Cancer Cell Biology, Nagoya University School of Medicine, Japan.
Kojima
K
K
Hanaichi
T
T
Masaki
S
S
Suzuki
M
M
Umekawa
H
H
Yoshida
S
S
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D019915:DNA Primase; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008970:Molecular Weight; D011487:Protein Conformation; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D013950:Thymus Gland / Q000201:enzymology*
D046911:Macromolecular Substances; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases