Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmc1 homolog, in Pyrococcus furiosus.
Pyrococcus furiosus has an operon containing the DNA polymerase II (PolD) gene and three other genes. Using a two-hybrid screening to examine the interactions of the proteins encoded by the operon, we identified a specific interaction between the second subunit of PolD (DP1) and a Rad51/Dmc1 homologous protein (RadB). To ensure the specific interaction between these two proteins, each gene in the operon was expressed in Escherichia coli or insect cells separately and the products were purified. The in vitro analyses using the purified proteins also showed the interaction between DP1 and RadB. The deletion mutant analysis of DP1 revealed that a region important for binding with RadB is located in the central part of the sequence (amino acid residues 206-498). This region has an overlap to the C-terminal half (amino acids 334-613), which is highly conserved among euryarchaeal DP1s and is essential for the activity of PolD. Our results suggest that, although RadB does not noticeably affect the primer extension ability of PolD in vitro, PolD may utilize the RadB protein in DNA synthesis under certain conditions.
Hayashi
I
I
Department of Molecular Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan.
Morikawa
K
K
Ishino
Y
Y
D000818:Animals; D019843:Archaeal Proteins; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D004257:DNA Polymerase II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry; D007313:Insecta; D008969:Molecular Sequence Data; D009876:Operon; D016133:Polymerase Chain Reaction; D020836:Protein Structure, Quaternary; D020027:Pyrococcus furiosus / Q000235:genetics* / Q000378:metabolism*; D051135:Rad51 Recombinase; D011938:Rec A Recombinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection
D019843:Archaeal Proteins; D018797:Cell Cycle Proteins; C513033:DMC1 protein, S cerevisiae; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C403141:RadB protein, Pyrococcus furiosus; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D011938:Rec A Recombinases; D004257:DNA Polymerase II