Both RadA and RadB are involved in homologous recombination in Pyrococcus furiosus.
RecA and Rad51 proteins are essential for homologous recombination in Bacteria and Eukarya, respectively. Homologous proteins, called RadA, have been described for Archaea. Here we present the characterization of two RecA/Rad51 family proteins, RadA and RadB, from Pyrococcus furiosus. The radA and radB genes were not induced by DNA damage resulting from exposure of the cells to gamma and UV irradiation and heat shock, suggesting that they might be constitutively expressed in this hyperthermophile. RadA had DNA-dependent ATPase, D-loop formation, and strand exchange activities. In contrast, RadB had a very weak ATPase activity that is not stimulated by DNA. This protein had a strong binding affinity for DNA, but little strand exchange activity could be detected. A direct interaction between RadA and RadB was detected by an immunoprecipitation assay. Moreover, RadB, but not RadA, coprecipitated with Hjc, a Holliday junction resolvase found in P. furiosus, in the absence of ATP. This interaction was suppressed in the presence of ATP. The Holliday junction cleavage activity of Hjc was inhibited by RadB in the absence, but not in the presence, of ATP. These results suggest that RadB has important roles in homologous recombination in Archaea and may regulate the cleavage reactions of the branch-structured DNA.
Komori
K
K
Departments of Molecular Biology and Structural Biology, Biomolecular Engineering Research Institute, Suita, Osaka 565-0874, Japan.
Miyata
T
T
DiRuggiero
J
J
Holley-Shanks
R
R
Hayashi
I
I
Cann
I K
IK
Mayanagi
K
K
Shinagawa
H
H
Ishino
Y
Y
D000251:Adenosine Triphosphatases / Q000378:metabolism; D019843:Archaeal Proteins; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D020027:Pyrococcus furiosus / Q000235:genetics*; D011995:Recombination, Genetic
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; C117252:RadA protein, archaeal; C403141:RadB protein, Pyrococcus furiosus; D004247:DNA; D000251:Adenosine Triphosphatases