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Nucleic Acids Research, 1985, Vol. 13, No. 18 6635-6649
© 1985

Articles

Characterization of a Mr= 56,000 polypeptide associated with 10S DNA polymerase {alpha} purified from calf thymus using monoclonal antibody

Shigeo Masaki*, Katsuyuki Tamai1, Rika Suzuki, Kazushi Tanabe2, Taijo Takahashi2 and Shonen Yoshida

Department of Biochemistry, Institute for Developmental Research Aichi Prefecture Colony, Kamiyacho, Kasugai, Aichi 480-03 1Ina Research Division, Medical and Biological Laboratories, Ltd. Ohara, Terasawaoka, Ina, Nagano 396 2Laboratory of Biochemistry, Aichi Cancer Center Research Institute Chikusa-ku, Nagoya, Aichi 464, Japan

*To whom correspondence should be addressed.

Received May 7, 1985. Revised August 12, 1985. Accepted August 23, 1985.

Existence of a Mr= 56,000 polypeptide associated with 10S DNA polymerase {alpha} was shown by production of a monoclonal anti-calf thymus 10S DNA polymerase {alpha} antibody secreted from a hybridoma line named 3H1. The antibody bound three polypeptides with Mr= 180,000, 56,000 and 32,000 in hydroxylapatite fraction of 10S DNA polymerase {alpha} by immunoblot. The antibody co-precipitated the polypeptides with the large polypeptide (Mr= 150,000–140,000) of 10S DNA polymerase {alpha} with the aid of second antibody. Among three polypeptides, the Mr= 56,000 polypeptide was co-purified with DNA polymerase {alpha} through DNA-cellulose chromatography and repeated sucrose rate-zonal centrifugations. The Mr= 56,000 polypeptide was still associated with 10S DNA polymerase {alpha} after second sucrose rate-zonal centrifugation, but the amount of it was reduced. The polypeptide was banded at pH 7.2–8.0 and displayed microheterogeneity in respect of isoelectric point by isoelectrofocusing with 7 M urea, and showed weak DNA-binding property after blotting onto a nitrocellulose. The antibody against the polypeptide precipitated DNA polymerase {alpha} from human, rat, and mouse, and Mr= 56,000 and 32,000 polypeptides were detected in these DNA polymerase {alpha} fractions by immunoblot. These results suggest that the polypeptide with Mr= 56,000 may take part in the DNA polymerase reaction.


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