Related Experiment Videos
A bacteriophage-induced 5-methyldeoxycytidine 5'-monophosphate kinase
Abstract:
Bacteriophage XP-12-infected Xanthomonas oryzae have been found to be a source of a kinase preparation which converts m5dCMP to m5dCDP and then to m5dCTP using ATP as the phosphate donor. Optimal formation of the triphosphate required the presence of creatine phosphate and creatine kinase. In the presence of dGTP, dTTP and dATP, Escherichia coli DNA polymerase I and T4 DNA polymerase catalyzed the incorporation of m5dCTP into DNA just as efficiently as that of dCTP. Neither dTMP nor dCMP served as substrate for the m5dCMP monophosphate kinase. Analogous preparations from uninfected X. oryzae were unable to phosphorylate m5dCMP.
Insights
Bacteriophage XP-12 infection of Xanthomonas oryzae yields a kinase that converts m5dCMP to m5dCTP. This modified nucleotide is efficiently incorporated into DNA by DNA polymerases.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Modified nucleosides in DNA can arise from various biological processes.
- Bacteriophages are viruses that infect bacteria and can alter their host's metabolic pathways.
Purpose of the Study:
- To investigate the enzymatic conversion of m5dCMP to its triphosphate form.
- To determine if m5dCTP can be incorporated into DNA by bacterial DNA polymerases.
Main Methods:
- Preparation of kinase from bacteriophage XP-12 infected Xanthomonas oryzae.
- Enzymatic assays to measure phosphorylation of m5dCMP to m5dCDP and m5dCTP.
- DNA synthesis assays using E. coli DNA polymerase I and T4 DNA polymerase with m5dCTP.
Main Results:
- A kinase preparation from infected X. oryzae efficiently converted m5dCMP to m5dCDP and m5dCTP using ATP.
- Optimal triphosphate formation required creatine phosphate and creatine kinase.
- Escherichia coli DNA polymerase I and T4 DNA polymerase incorporated m5dCTP into DNA as efficiently as dCTP.
- Uninfected X. oryzae preparations could not phosphorylate m5dCMP.
Conclusions:
- Bacteriophage XP-12 infection induces enzymes capable of synthesizing m5dCTP.
- m5dCTP is a viable substrate for DNA polymerases, suggesting potential roles in DNA replication or repair.