DNA polymerase-alpha. Common polypeptide core structure of three enzyme forms from human KB cells

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Human KB cells exhibit multiple DNA polymerase-alpha activities.
• A predominant cytoplasmic form has been previously characterized.
• Minor cytoplasmic and nuclear DNA polymerase-alpha forms were identified.

Purpose of the Study:

• To purify and structurally characterize the minor cytoplasmic and nuclear DNA polymerase-alpha species.
• To compare the structures of all identified KB cell DNA polymerase-alpha forms.
• To elucidate the subunit composition of these enzymes.

Main Methods:

• Enzyme purification to near-homogeneity.
• Physicochemical characterization (e.g., sedimentation analysis).
• High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography for subunit analysis.

Main Results:

• Purification of two minor DNA polymerase-alpha species.
• All three forms are 7 S species with a 140,000 molecular weight protomer.
• Similar subunit structures composed of polypeptides (70,000, 65,000, 59,000, 55,000 daltons) were observed.
• No polypeptides larger than 70,000 daltons were detected.

Conclusions:

• The three KB cell DNA polymerase-alpha forms are structurally and enzymologically similar.
• The observed polypeptide composition suggests microheterogeneity possibly involving two primary species.
• This study provides the first comparative structural analysis of multiple DNA polymerase-alpha forms from a single source and characterizes a nuclear DNA polymerase-alpha.