Both RadA and RadB Are Involved in Homologous Recombination inPyrococcus furiosus

Abstract

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 ␥ and UV irradiation and heat shock, suggesting that they might be constitutively expressed in this hyperthermophile. RadA had DNA-dependent ATPase, Dloop 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.Genetic recombination is important both in generating genetic diversity and in repairing DNA damages. Homologous DNA recombination involves multistep reactions. The molecular mechanisms of the early stage include pairing and strand exchange reactions of two homologous DNA strands. The RecA/ Rad51 family proteins have a central role in the initiation step by binding to single-stranded DNA (ssDNA), 1 which results in the formation of a helical nucleoprotein filament (reviewed in Refs. 1 and 2). RecA proteins of eubacteria and Rad51 proteins of eukaryotes have been extensively studied to date (reviewed in Refs. 3-5). RecA/Rad51 structural homologs have also been found in the Archaea and named RadA (6 -10). The amino acid sequences of archaeal RadAs are much more similar to those of eukaryotic Rad51 homologs than to those of bacterial RecA homologs. Preliminary characterization shows that the archaeal RadAs found in Sulfolobus solfataricus, Desulfurococcus amylolyticus, and Pyrobaculum islandicum are functionally similar to the RecA/Rad51 family proteins found in the other domains (11-14), and they are now thought to play a critical role in recombination and repair in Archaea.To understand the detailed mechanism of the DNA recombination in Archaea, we have been investigating the proteins related to this process from the hyperthermophilic archaeon, Pyrococcus furiosus (15). We identified a Rad51-like protein that is encoded in an operon that includes a novel heterodimeric DNA polymerase (polymerase II or D) and an Orc1 (origin recognition complex protein 1)-like protein (9). When we first reported this operon, we called the Rad51-like protein RadA. However, a subsequent report identified two Rad51/ Dmc1 homologs in the P. ...

An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

“…The archaeal homolog of RecA, Sulfolobus solfataricus RadA protein, can form octameric rings or helical filaments (40). The Pyrococcus furiosus RadA protein (PfRad51) was crystallized as a heptamer (41), and EM studies revealed hexameric filaments (42). The human Dmc1 protein is a RecA homolog that catalyzes homologous pairing during meiosis.…”

Domain Analysis of an Archaeal RadA Protein for the Strand Exchange Activity

“…We cloned the two genes, radA and radB from the hyperthermophilic archaeon, Pyrococcus furiosus, expressed them in Escherichia coli, and characterized the gene products, RadA and RadB (30). The amino acid sequences of RadA and RadB are very similar to that of eukaryotic Rad51 (54 and 29% similarity, respectively, to S. cerevisiae Rad51).…”

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.purification procedure of the RadA protein are described in the accompanying paper (30). The N-terminal-and C-terminal truncated RadAs (RadA-n, Met 1 -Arg…”

“…Nuclease assays were done to check the contamination of any deoxyribonuclease in the purified proteins using 32 P-labeled DNA (both single-stranded and double-stranded) as described (30). To reconstitute RadA-n and RadA-c, both proteins were mixed with the various molar ratio and were incubated at 60°C for 3 min before use for various assays described below.…”

Origins and evolution of the recA / RAD51 gene family: Evidence for ancient gene duplication and endosymbiotic gene transfer

“…In eubacteria, only one recA gene has been previously reported in each species (7). Unlike eubacteria, several archaeal species have two recA͞RAD51-like genes, called RADA and RADB (Table 1, which is published as supporting information on the PNAS web site) (8,9). Among eukaryotes, the budding yeast and the fission yeast Schizosaccharomyces pombe contain four RAD51-like genes (RAD51, DMC1, RAD55͞rhp55, and RAD57͞rhp57) (5,6,10,11).…”