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On the DNA polymerase III of mouse myeloma: partial purification and characterization
Abstract:
A high molecular weight membrane-bound DNA polymerase from the mouse myeloma, MOPC-104E, has been purified extensively, and characterized with regard to physical and reaction properties. This enzyme, which is readily distinguishable from other myeloma enzymes that are analogous to the recognized forms of cellular DNA polymerase, is ddesignated DNA polymerase III. DNA polymerase III activity in whole homogenates from MOPC-104E was solubilized and then prurifed using a series of ion-exchange chromatographic procedures followed by DNA-cellulose chromatography and glycerol gradient centrifugation; the enzyme activity as measured with poly(rA)-(dT)12-18 as template-primer and Mn2+ as divalent cation, was purified as much as 18,000-fold. In the final stages of the pruification, DNA polymerase III possessed no detectable RNA polymerase activity, nucleoside diphosphokinase activity, or nucease activity toward DNA or single- and double-stranded RNA...
Insights
Researchers purified a novel mouse myeloma enzyme, DNA polymerase III, distinguishing it from other cellular DNA polymerases. This high molecular weight enzyme shows specific activity with a synthetic template-primer, indicating its unique role.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Cellular DNA polymerases are crucial for DNA replication and repair.
- Mouse myeloma cell lines provide a source for studying novel enzymes.
- Previous research identified various DNA polymerase forms in mammalian cells.
Purpose of the Study:
- To extensively purify and characterize a high molecular weight, membrane-bound DNA polymerase from MOPC-104E mouse myeloma.
- To distinguish this novel enzyme, designated DNA polymerase III, from known cellular DNA polymerases.
- To assess the physical and reaction properties of the purified enzyme.
Main Methods:
- Solubilization of DNA polymerase III activity from MOPC-104E homogenates.
- Purification using sequential ion-exchange chromatography, DNA-cellulose chromatography, and glycerol gradient centrifugation.
- Enzyme activity assay using poly(rA)-(dT)12-18 template-primer with Mn2+ as the divalent cation.
Main Results:
- Achieved an 18,000-fold purification of DNA polymerase III.
- Demonstrated that the purified enzyme is distinct from other known myeloma enzymes.
- Confirmed the absence of RNA polymerase, nucleoside diphosphokinase, and nuclease activities in the final purified enzyme preparation.
Conclusions:
- Successfully purified and characterized a unique DNA polymerase III from mouse myeloma MOPC-104E.
- DNA polymerase III exhibits distinct properties differentiating it from other cellular DNA polymerases.
- The purified enzyme is a specific DNA polymerase with no detectable contaminating enzymatic activities.