A novel DNA polymerase in the hyperthermophilic archaeon, <i>Pyrococcus furiosus</i>: gene cloning, expression, and characterization

We also constructed and analyzed three internal deletion mutants and two site-directed mutants in the region of the putative zinc finger motif (cysteine cluster II) of DP2Pho at the COOH-terminal. We found that the internal region of the zinc finger motif is critical for the 3-5 exonuclease, but is dispensable for the DNA polymerization.Family D DNA polymerase (PolD) 1 is a recently found DNA polymerase that exists extensively in Euryarchaeota of Archaea, the third domain of life (1, 2 PolD is composed of a small subunit (DP1) and a large subunit (DP2). The interaction of DP1 and DP2 is essential for the stability and full activity of the enzyme (15, 16). DP1 or DP2 expressed in Escherichia coli is unstable and easily degraded. PolD possesses strong DNA polymerizing and 3Ј-5Ј exonuclease (proofreading) activities like other DNA replication polymerases, however, common motifs for the catalysis of other DNA polymerases are absent or not functional in PolD (17,19). Two invariant residues, Asp 1122 and Asp 1124 of DP2Pho, located in the most conserved region in the large subunit of PolD, were identified to be the catalytic residues for the DNA polymerization activities (17), indicating that the COOH-terminal of DP2 is involved in the formation of the catalytic center for DNA synthesis. A heterotetrameric structure for PolD from P. horikoshii (PolDPho) was proposed based on the result of gel filtration (17).Several lines of evidence indicate that family D DNA polymerase is likely the major DNA polymerase or replicase in Euryarchaeota, participating in DNA replication, repair, and recombination. In the genomes of the genus Pyrococcus, the genes coding the two subunits of PolD are adjacent to the replication origin and several essential genes for DNA metabolism (20). DP1 shows low but significant homology to the small subunit of eukaryote DNA polymerase ␣, ␦, and ⑀ (19, 21), and contains domain and catalytic motifs for the 3Ј-5Ј exonuclease, similar to those of Mre11, a nuclease involved in double strand DNA break repair (18,21,22). The amino acid sequences of DP2 contain a short region at the COOH-terminal, which shows homology to the catalytic subunit of yeast DNA polymerase ⑀ (this paper) around the COOH-terminal putative zinc finger motif (cysteine cluster II). This homologous region in yeast polymerase ⑀ is found to be vital for the survival of yeast (23,24). PolD interacts with many proteins related to DNA replication, repair, and recombination. It was found that DP1 from P. furiosus interacts specifically with archaeal RadB by yeast two-hybrid analysis and in vitro pull-down assays (25). DP2 directly interacts with proliferating cell nuclear antigen as * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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confidence: 99%

“…The interaction of DP1 and DP2 is essential for the stability and full activity of the enzyme (15,16). DP1 or DP2 expressed in Escherichia coli is unstable and easily degraded.…”

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confidence: 99%

Subunit Interaction and Regulation of Activity through Terminal Domains of the Family D DNA Polymerase from Pyrococcus horikoshii

Shen

Tang

Matsui

2003

“…[1][2][3][4]. Two genes, radA and radB, both of which encode RecA/Rad51 family proteins, have been found in the genomes of archaeal organisms (5,6). All of the euryarchaeotic total genome sequences that have been determined to date contain both RadA and RadB (7,8).…”

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confidence: 99%

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

Komori¹,

Miyata²,

Daiyasu³

et al. 2000

Self Cite

Archaeal RadA, like eukaryotic Rad51 and bacterial RecA, promotes strand exchange between DNA strands with homologous sequences in vitro and is believed to participate in the homologous recombination in cells. The amino acid sequences of the archaeal RadA proteins are more similar to the eukaryotic Rad51s rather than the bacterial RecAs, and the N-terminal region containing domain I is conserved among the RadA and Rad51 proteins but is absent from RecA. To understand the structure-function relationship of RadA, we divided the RadA protein from Pyrococcus furiosus into two parts, the N-terminal one-third (RadA-n) and the residual Cterminal two-thirds (RadA-c), the latter of which contains the central core domain (domain II) of the RecA/ Rad51 family proteins. RadA-c had the DNA-dependent ATPase activity and the strand exchange activity by itself, although much weaker (10%) than that of the intact RadA. These activities of RadA-c were restored to 60% of those of RadA by addition of RadA-n, indicating that the proper active structure of RadA was reconstituted in vitro. These results suggest that the basic activities of the RecA/Rad51 family proteins for homologous recombination are derived from domain II, and the Nterminal region may help to enhance the catalytic efficiencies.Genetic recombination is important in generating genetic diversity and in repairing DNA damage. The RecA protein of eubacteria and the Rad51 protein of eukaryotes play a central role in homologous DNA recombination by searching for sequence homology and exchanging the DNA strands (reviewed in Refs.

“…We characterized the Pyrococcus furiosus RadA and RadB proteins (13)(14)(15), which play a central role in the initiation of homologous recombination. These archaeal recombinases have sequences more similar to that of eukaryotic Rad51 than bacterial RecA (16,17). In addition, we showed that the P. furiosus RPA, which is composed of three subunits, RPA41, RPA14, and RPA32, like the eukaryotic RPA (p70-p14-p32), but different from bacterial SSB (homotetramer), clearly stimulated a RadA-mediated strand exchange reaction (18).…”

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confidence: 96%

Identification of a Novel Helicase Activity Unwinding Branched DNAs from the Hyperthermophilic Archaeon, Pyrococcus furiosus

Fujikane

Komori

Shinagawa

et al. 2005

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To identify the branch migration activity in archaea, we fractionated Pyrococcus furiosus cell extracts by several chromatography and assayed for ATP-dependent resolution of synthetic Holliday junctions. The target activity was identified in the column fractions, and the optimal reaction conditions for the branch migration activity were determined using the partially purified fraction. We successfully cloned the corresponding gene by screening a heat-stable protein library made by P. furiosus genomic DNA. The gene, hjm (Holliday junction migration), encodes a protein composed of 720 amino acids. The Hjm protein is conserved in Archaea and belongs to the helicase superfamily 2. A homology search revealed that Hjm shares sequence similarity with the human Pol⌰, HEL308, and Drosophila Mus308 proteins, which are involved in a DNA repair, whereas no similar sequences were found in bacteria and yeast. The Hjm helicase may play a central role in the repair systems of organisms living in extreme environments.Homologous recombination plays important roles in DNA metabolisms, including several DNA repair processes and the generation of genetic diversity in living cells. The Holliday junction (HJ) 1 is an important intermediate during the homologous recombination process (1). The molecular mechanism of the early stage of homologous recombination has been extensively investigated, and the proteins involved in this process are conserved in the three biological domains: Bacteria, Eukarya, and Archaea (2, 3). On the other hand, the protein factors involved in the late stage of the process have been identified only in Bacteria, in which the RuvABC-Holliday junction complex processes the recombinational intermediates (4, 5). In Eukarya, the activities for branch migration and resolution of the HJ have been reported (6 -9); however, the corresponding proteins have yet to be identified. A recent report showed that RAD51C, a RAD51 paralog in human cells, is involved in HJ processing (10).Archaea, the third domain of life (11), is distinct from both Bacteria and Eukarya. Archaea share many similarities with Eukarya in their genetic information processing pathways, including DNA replication, transcription, and translation (12), although cellular structure is prokaryotic. Therefore, the archaeal processes provide a useful model systems to understand the much more complex mechanisms of their eukaryotic equivalents. Proteins involved in homologous recombination are also conserved between Archaea and Eukarya. We characterized the Pyrococcus furiosus RadA and RadB proteins (13-15), which play a central role in the initiation of homologous recombination. These archaeal recombinases have sequences more similar to that of eukaryotic Rad51 than bacterial RecA (16,17). In addition, we showed that the P. furiosus RPA, which is composed of three subunits, RPA41, RPA14, and RPA32, like the eukaryotic RPA (p70-p14-p32), but different from bacterial SSB (homotetramer), clearly stimulated a RadA-mediated strand exchange reaction (18). The homologs...