Structural relationships between two forms of DNA polymerase epsilon from calf thymus.
We previously reported purification of two forms of DNA polymerase epsilon from calf thymus (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). We have now used the "polymerase trap" photolabeling method to identify the polypeptides containing the polymerase active site in each enzyme preparation. The molecular mass of these polypeptides are 210 and 145 kDa for the polymerases now designated epsilon and epsilon*, respectively. Renaturation of polymerase activity from denaturing gel electrophoresis corroborates the polymerase trap results. Photolabeling of polymerase fractions suggests that the smaller subunit is derived by proteolysis of the larger subunit during purification. Native sedimentation coefficient measurements of polymerase-containing column fractions further suggest a precursor/product relationship between the two polymerases. Response of polymerization activity to a battery of inhibitors normally used to distinguish mammalian nuclear DNA polymerases was found to be essentially identical for polymerases epsilon, epsilon*, and the epsilon* generated in fractions initially containing epsilon. These latter results demonstrate that the loss of the protease-sensitive domain of the active site subunit does not affect catalytic function as measured in a standard DNA polymerase assay. The sole apparent functional difference observed here between the epsilon and epsilon* forms is evidence that only the full-length epsilon form can be directly photocrosslinked to dATP, independent of DNA synthesis. Photolabeling of the post-microsomal supernatant fraction from thymus glands obtained from fetal calves reveals the presence of both the epsilon and epsilon* polypeptide.
Siegal
G
G
Department of Biochemistry and Cancer Center, University of Rochester School of Medicine and Dentistry, New York 14642.
Turchi
J J
JJ
Jessee
C B
CB
Mallaber
L M
LM
Bambara
R A
RA
Myers
T W
TW
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019384:Nucleic Acid Synthesis Inhibitors; D010777:Photochemistry; D011489:Protein Denaturation; D013950:Thymus Gland / Q000201:enzymology*
D003432:Cross-Linking Reagents; D007527:Isoenzymes; D019384:Nucleic Acid Synthesis Inhibitors; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase