A RepA-like protein from bacteriophage BFK20 is a multifunctional protein with primase, polymerase, NTPase and helicase activities.
Phage BFK20 replication protein gp43 comprises an N-terminal prim-pol domain and a C-terminal domain similar to replicative helicases. We prepared and studied two recombinant proteins: gp43-1 containing both prim-pol and helicase domains, and gp43C with the helicase domain only. A SEC-MALS analysis indicated that gp43-1 forms a hexameric homooligomer. NTPase activity testing revealed that gp43-1 was able to hydrolyze a wide spectrum of NTPs, ATP the most efficiently. The ATPase activity of gp43-1 was strongly dependent on the presence of ssDNA in the reaction, but was low in the presence of dsDNA and in the absence of DNA. On the other hand, the ATPase activity of gp43C was very low regardless of the presence of DNA. The helicase activity of gp43-1 was detected using a fluorescence-based assay with a forked DNA substrate in the presence of ATP. However, no helicase activity could be detected for gp43C. We therefore conclude that the prim-pol domain is essential for the helicase and ssDNA-dependent ATPase activity of gp43-1.
Department of Genomics and Biotechnology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovakia.
Nora
N
Halgasova
Department of Genomics and Biotechnology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovakia.
Barbora
B
Solteszova
Department of Biochemistry and Structural Biology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovakia.
Vladimír
V
Pevala
Department for Structural and Computational Biology, Max F. Perutz Laboratories, Dr. Bohr-Gasse 9 (VBC 5), 1030 Vienna, Austria.
Július
J
Košťan
Department of Biochemistry and Structural Biology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovakia.
Eva
E
Kutejová
Department of Genomics and Biotechnology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovakia. Electronic address: Gabriela.Bukovska@savba.sk.
Gabriela
G
Bukovska
D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics*; D040142:Brevibacterium flavum / Q000821:virology; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D043583:Nucleoside-Triphosphatase / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*
D015534:Trans-Activators; C045264:replication initiator protein; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase; D043583:Nucleoside-Triphosphatase; D004265:DNA Helicases
Bacteriophage; Corynebacterium; DNA replication; Helicase; Protein expression; Replication protein