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Nucleoside diphosphokinase activity associated with DNA polymerases
Abstract:
Nucleoside diphosphokinase activity is present in highly purified preparations of DNA polymerase from Micrococcus luteus and Escherichia coli, and in a partially purified DNA polymerase from avian myeloblastosis virus. The activity is also observed in the protein fragment of molecular weight 76,000 that is produced by subtilisin cleavage of DNA polymerase I from E. coli. The NDP kinase activity in DNA polymerase preparations from M. luteus uses various ribo- and deoxyribonucleoside di- and triphosphates as substrates. The presence of this activity in preparations of DNA polymerase results in the apparent use of deoxyribonucleoside diphosphates as substrates for DNA synthesis, provided that some triphosphate is present to serve as a phosphate donor.
Insights
Nucleoside diphosphokinase (NDP kinase) activity was found in DNA polymerase preparations from bacteria and viruses. This enzyme activity can lead to the apparent use of diphosphates in DNA synthesis when triphosphates are present.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- DNA polymerases are crucial enzymes for DNA replication and repair.
- Nucleoside diphosphokinases (NDP kinases) are enzymes that catalyze the transfer of a phosphate group from nucleoside triphosphates to nucleoside diphosphates.
- The presence of enzymatic activities other than DNA polymerization in purified DNA polymerase preparations can complicate the interpretation of experimental results.
Purpose of the Study:
- To investigate the presence and nature of nucleoside diphosphokinase (NDP kinase) activity in purified DNA polymerase preparations.
- To determine if NDP kinase activity affects the apparent substrate utilization of DNA polymerases.
- To characterize the NDP kinase activity associated with DNA polymerase I from Escherichia coli.
Main Methods:
- Purification of DNA polymerase enzymes from Micrococcus luteus, Escherichia coli, and avian myeloblastosis virus.
- Enzymatic assays to detect and characterize NDP kinase activity using various ribo- and deoxyribonucleoside di- and triphosphates.
- Subtilisin cleavage of E. coli DNA polymerase I to identify the fragment responsible for NDP kinase activity.
Main Results:
- Highly purified DNA polymerase preparations from M. luteus and E. coli, as well as partially purified avian myeloblastosis virus DNA polymerase, exhibited significant NDP kinase activity.
- NDP kinase activity was also detected in a 76,000 molecular weight protein fragment of E. coli DNA polymerase I, generated by subtilisin cleavage.
- The NDP kinase activity in M. luteus DNA polymerase preparations utilized a broad range of ribonucleoside and deoxyribonucleoside di- and triphosphates as substrates.
- The presence of NDP kinase activity led to the apparent incorporation of deoxyribonucleoside diphosphates into DNA, provided a triphosphate was available as a phosphate donor.
Conclusions:
- DNA polymerase preparations can contain associated NDP kinase activity.
- This associated NDP kinase activity can influence the interpretation of DNA synthesis assays by causing the apparent utilization of diphosphates.
- The findings highlight the importance of assessing enzyme purity and potential contaminating activities in biochemical studies of DNA polymerases.