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. ...

We describe here a DNA polymerase family highly conserved in Euryarchaeota, a subdomain of Archaea. The DNA polymerase is composed of two proteins, DP1 and DP2. Sequence analysis showed that considerable similarity exists between DP1 and the second subunit of eukaryotic DNA polymerase ␦, a protein essential for the propagation of Eukarya, and that DP2 has conserved motifs found in proteins with nucleotide-polymerizing activity. These results, together with our previous biochemical analyses of one of the members, DNA polymerase II (DP1 ؉ DP2) from Pyrococcus furiosus, implicate the DNA polymerases of this family in the DNA replication process of Euryarchaeota. The discovery of this DNA-polymerase family, aside from providing an opportunity to enhance our knowledge of the evolution of DNA polymerases, is a significant step toward the complete understanding of DNA replication across the three domains of life.The DNA replication apparatus has been well characterized in Bacteria, with Escherichia coli serving as a model (1, 2). In this organism, chromosomal duplication is the function of the DNA polymerase III holoenzyme. The genes encoding all 10 subunits of the holoenzyme have been identified, and these proteins have been overproduced, purified, and reconstituted. Proteins with corresponding functions have been identified in Eukarya (3, 4). However, in both Bacteria and Eukarya, few of these similarly functioning proteins exhibit any meaningful amino acid conservation.Two decades ago, biologists witnessed a landmark discovery by Woese and Fox (5), who announced the existence of a third form of life, currently referred to as Archaea (6). Even though members of this domain are dissimilar to the eukaryotes (6), archaeal information-processing systems (i.e., transcription, translation, and apparently replication systems) are more similar to the eukaryotic than to the bacterial versions. The study of archaeal information processing may, therefore, help us to understand the structure, function, and evolution of homologous eukaryotic systems and vice versa.Previously, we cloned a DNA-polymerase gene that encodes an eukaryote-like family B (␣-like) DNA polymerase from the euryarchaeote Pyrococcus furiosus (7). Furthermore, we showed that the crenarchaeote Pyrodictium occultum possesses at least two family B DNA polymerases (8). Including our results, every archaeal DNA polymerase sequenced before the first complete archaeal genome report of Methanococcus jannaschii (9) was a single-subunit member of family B (10-13).An extremely puzzling observation from the complete genome sequence of M. jannaschii was the presence of what is apparently a single DNA polymerase sequence (9). This finding was inconsistent with the presence of multiple DNA polymerases serving different functions in other forms of life. In a recent report, Olsen and Woese (14) noted the possibility that the archaeal replicative polymerase may have eluded researchers. Edgell and Doolittle (15) also argued that nonhomologous proteins are likely recruite...

The Holliday junction is an essential intermediate of homologous recombination. RecA of Bacteria, Rad51 of Eukarya, and RadA of Archaea are structural and functional homologs. These proteins play a pivotal role in the formation of Holliday junctions from two homologous DNA duplexes. RuvC is a specific endonuclease that resolves Holliday junctions in Bacteria. A Holliday junction-resolving activity has been found in both yeast and mammalian cells. To examine whether the paradigm of homologous recombination apply to Archaea, we assayed and found the activity to resolve a synthetic Holliday junction in crude extract of Pyrococcus furiosus cells. The gene, hjc (Holliday junction cleavage), encodes a protein composed of 123 amino acids, whose sequence is not similar to that of any proteins with known function. However, all four archaea, whose total genome sequences have been published, have the homologous genes. The purified Hjc protein cleaved the recombination intermediates formed by RecA in vitro. These results support the notion that the formation and resolution of Holliday junction is the common mechanism of homologous recombination in the three domains of life.

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DNA and RNA frequently form various branched intermediates that are important for the transmission of genetic information. Helicases play pivotal roles in the processing of these transient intermediates during nucleic acid metabolism. The archaeal Hef helicase/ nuclease is a representative protein that processes flap- or fork-DNA structures, and, intriguingly, its C-terminal half belongs to the XPF/Mus81 nuclease family. Here, we report the crystal structure of the helicase domain of the Hef protein from Pyrococcus furiosus. The structure reveals a novel helical insertion between the two conserved helicase core domains. This positively charged extra region, structurally similar to the "thumb" domain of DNA polymerase, plays critical roles in fork recognition. The Hef helicase/nuclease exhibits sequence similarity to the Mph1 helicase from Saccharomyces cerevisiae; XPF/Rad1, involved in DNA repair; and a putative Hef homolog identified in mammals. Hence, our findings provide a structural basis for the functional mechanisms of this helicase/nuclease family.

Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32. The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M. jannaschii RPA. On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya. However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32. The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P. furiosus RadA protein. Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P. furiosus cells. These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P. furiosus.Single-stranded DNA-binding protein (SSB) 1 in Bacteria and replication protein A (RPA) in Eukarya play essential roles in DNA replication, recombination, and repair. (1-3). The bacterial SSB and the eukaryotic RPA bind to single-stranded DNA as a homotetramer and a heterotrimer, respectively. Although there is little amino acid sequence similarity between SSB and RPA, recent analyses of the three-dimensional structures revealed that these proteins have a structurally similar domain (4 -6) containing a common fold, called the oligonucleotide/ oligosaccharide binding (OB) fold (7). The presence of this common structure in SSB and RPA, and also in the RNA binding domain of Escherichia coli polynucleotide phosphorylase (8), suggests that the mechanism of single-stranded nucleic acid binding appears to be conserved in the biological domains.Bacterial SSB is a homotetramer of a 20-kDa peptide with one OB fold. In contrast, eukaryotic RPA is a stable heterotrimer composed of 70, 32, and 14 kDa subunits. The largest subunit, RPA70, contains two tandem repeats of an OB fold that are responsible for the major interaction with a singlestranded DNA in the central region (5, 9 -11). The N-terminal and C-terminal regions of RPA70 mediate interactions with many cellular or viral proteins and RPA32, respectively (12, 13). The zinc finger motif, which is important for RPA function, is strongly conserved in the C-terminal region of RPA70 (14, 15). The middle subunit, RPA32, contains an OB fold at the central region (6,16,17). The RPA32 interacts with other RPA subunits and various cellular proteins at the C-...

The architectural similarity of Hjc to restriction endonucleases allowed us to construct a putative model of the complex with Holliday junction. This model accounts for how Hjc recognizes and resolves the junction DNA in a specific manner. Mutational and biochemical analyses highlight the importance of some loops and the amino terminal region in interaction with DNA.

We identified a novel structure-specific endonuclease in Pyrococcus furiosus. This nuclease contains two distinct domains, which are similar to the DEAH helicase family at the N-terminal two-third and the XPF endonuclease superfamily at the C-terminal one-third of the protein, respectively. The C-terminal domain has an endonuclease activity cleaving the DNA strand at the 5'-side of nicked or flapped positions in the duplex DNA. The nuclease also incises in the proximity of the 5'-side of a branch point in the template strand for leading synthesis in the fork-structured DNA. The N-terminal helicase may work cooperatively to change the fork structure suitable for cleavage by the C-terminal endonuclease. This protein, designated as Hef (helicase-associated endonuclease for fork-structured DNA), may be a prototypical enzyme for resolving stalled forks during DNA replication, as well as working at nucleotide excision repair.