title: Identification of a bifunctional primase-polymerase domain of corynephage BFK20 replication protein gp43.
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.
authors: Department of Genomics and Biotechnology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovakia.
authors: Nora
authors: N
authors: Halgasova
authors: Irena
authors: I
authors: Mesarosova
authors: Gabriela
authors: G
authors: Bukovska
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000821:virology*; D019915:DNA Primase / Q000235:genetics* / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D010710:Phosphates; D011994:Recombinant Proteins; D014764:Viral Proteins; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase