Related Experiment Videos
Structural relationships between two forms of DNA polymerase epsilon from calf thymus
G Siegal1, J J Turchi, C B Jessee
1Department of Biochemistry and Cancer Center, University of Rochester School of Medicine and Dentistry, New York 14642.
The Journal of Biological Chemistry
|February 25, 1992
Summary
Researchers identified two forms of DNA polymerase epsilon (epsilon and epsilon*) in calf thymus. The smaller epsilon* form appears to be a proteolyzed version of the larger epsilon form, with similar catalytic activity but distinct dATP binding properties.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Two forms of DNA polymerase epsilon were previously purified from calf thymus.
- The relationship between these two forms and their active site components required further investigation.
Purpose of the Study:
- To identify the polypeptides containing the active site of DNA polymerase epsilon and epsilon* using the polymerase trap photolabeling method.
- To investigate the potential precursor-product relationship between the two polymerase forms.
- To assess the functional differences between DNA polymerase epsilon and epsilon*.
Main Methods:
- Utilized the "polymerase trap" photolabeling technique to identify active site polypeptides.
- Performed renaturation of polymerase activity from denaturing gel electrophoresis.
- Conducted native sedimentation coefficient measurements.
- Assessed enzyme activity using a battery of mammalian nuclear DNA polymerase inhibitors.
- Investigated photocrosslinking to dATP and DNA synthesis.
Main Results:
- Identified 210 kDa (epsilon) and 145 kDa (epsilon*) polypeptides as containing the polymerase active site.
- Evidence suggests epsilon* is derived from epsilon via proteolysis during purification.
- Both forms exhibit identical responses to a range of polymerase inhibitors, indicating conserved catalytic function.
- Only the full-length epsilon form can be directly photocrosslinked to dATP independently of DNA synthesis.
Conclusions:
- DNA polymerase epsilon* is likely a proteolyzed form of epsilon, with the loss of a protease-sensitive domain not affecting standard catalytic activity.
- A key functional difference lies in the ability of the full-length epsilon form to bind dATP directly.
- Both epsilon and epsilon* forms are present in fetal calf thymus supernatant, suggesting their biological relevance.