PMID:22108584

Citation	Halgasova, N, Mesarosova, I and Bukovska, G (2012) Identification of a bifunctional primase-polymerase domain of corynephage BFK20 replication protein gp43. Virus Res. 163:454-60
Abstract	Replication protein gp43 is a gene product of orf43, from the genome of corynephage BFK20 and carries two different domains. The C-terminal part of gp43 is similar to F4-type helicases and the N-terminal part resembles the rare primase-polymerase (prim-pol) domain. We expressed the 372 amino acids of the gp43 N-terminus in the pET expression system as recombinant protein gp43N with His-Tag fusion on both the N- and C-termini. The protein gp43N was purified by immobilized cobalt or nickel ion affinity chromatography. Gel filtration chromatography on Superose 12 showed that the purified protein elutes at an apparent molecular weight of 80 kDa, suggesting that it may be a dimer. We detected primase and DNA polymerase activities in gp43N using a simple method based on the determination of inorganic pyrophosphate and we demonstrated these two activities by polyacrylamide and agarose gel electrophoresis. In both primase and polymerase reactions, gp43N used only deoxyribonucleotides. By using defined single-stranded oligonucleotides as templates, we found that the primase is not highly sequence specific and does not require a specific trinucleotide for initiation of primer synthesis. The prim-pol domain of gp43 is the first such domain of a phage protein studied as an individual heterologous protein.
Links	PubMed Online version:10.1016/j.virusres.2011.11.005
Keywords	Amino Acid Sequence; Bacteriophages/enzymology; Bacteriophages/genetics; Chromatography, Affinity; Chromatography, Gel; Cloning, Molecular; Corynebacterium/virology; DNA Primase/genetics; DNA Primase/metabolism; DNA-Directed DNA Polymerase/genetics; DNA-Directed DNA Polymerase/metabolism; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Gene Expression; Molecular Sequence Data; Phosphates/metabolism; Protein Multimerization; Protein Structure, Tertiary; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Sequence Alignment; Substrate Specificity; Viral Proteins/genetics; Viral Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
9CAUD:Q3V5F2		GO:0003887: DNA-directed DNA polymerase activity	ECO:0001258: spectrophotometry evidence used in manual assertion		F		The gp43 protein from the bacteriophage BFK20 of the Brevibacterium flavum was suspected to exhibit polymerase activity. To purify samples of gp43, a plasmid that produces gp43 was transformed into E. coli, these cells were centrifuged, the supernatant was loaded onto a cobalt-affinity chromatography column, and the gp43 was collected from the column. The polymerase activity of gp43 was quantified by the amount of released free pyrophosphate through a colorimetric spectrophotometer analysis in vitro. The reaction mixture contained ssDNA and dNTP, and the addition of gp43 started the reaction with single-stranded DNA used as the template. Aliquots from the mixture were then added to thermostable inorganic pyrophosphatase.The phosphate that was released was quantified through a colorimetric spectrophotometer analysis, and results were shown in Figure 4. A negative control consisted of the reaction mixture without the thermostable inorganic pyrophosphatase. The negative control was subtracted from the experimental values to obtain the amount of phosphate released. From this procedure, researchers concluded that the gp43 protein was able to synthesize DNA.	complete CACAO 13720

References

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