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A Heterodimeric DNA Polymerase: Evidence That Members Of Proc. Natl. Acad. Sci. USA Vol. 95, pp. 14250–14255, November 1998 Evolution A heterodimeric DNA polymerase: Evidence that members of Euryarchaeota possess a distinct DNA polymerase (ArchaeayDNA replicationyDNA Polymerase dyhyperthermophileymethanogen) ISAAC K. O. CANN*, KAYOKO KOMORI*, HIROYUKI TOH†,SATORU KANAI†, AND YOSHIZUMI ISHINO*‡ Departments of *Molecular Biology and †Bioinformatics, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565, Japan Communicated by Carl R. Woese, University of Illinois at Urbana-Champaign, Urbana, IL, August 6, 1998 (received for review April 29, 1998) ABSTRACT We describe here a DNA polymerase family finding was inconsistent with the presence of multiple DNA highly conserved in Euryarchaeota, a subdomain of Archaea. polymerases serving different functions in other forms of life. The DNA polymerase is composed of two proteins, DP1 and In a recent report, Olsen and Woese (14) noted the possibility DP2. Sequence analysis showed that considerable similarity that the archaeal replicative polymerase may have eluded exists between DP1 and the second subunit of eukaryotic DNA researchers. Edgell and Doolittle (15) also argued that non- polymerase d, a protein essential for the propagation of homologous proteins are likely recruited into a replication Eukarya, and that DP2 has conserved motifs found in proteins function in one of the lineages, thereby replacing cenancestral with nucleotide-polymerizing activity. These results, together components. with our previous biochemical analyses of one of the members, We discovered a distinct DNA polymerase (Pol II) from a DNA polymerase II (DP1 1 DP2) from Pyrococcus furiosus, P. furiosus cell extract (16). During the purification of the implicate the DNA polymerases of this family in the DNA native Pol II from P. furiosus, deoxynucleotide incorporation replication process of Euryarchaeota. The discovery of this activity was detected from a protein (Pfu DP2) with an DNA-polymerase family, aside from providing an opportunity apparent molecular mass of 130 kDa and another protein with to enhance our knowledge of the evolution of DNA poly- a larger molecular mass, as determined by gel filtration. The merases, is a significant step toward the complete understand- presence of the larger protein prompted three hypotheses: (i) ing of DNA replication across the three domains of life. multimerization of the 130-kDa protein, (ii) an interaction of an accessory protein with the 130-kDa protein, and (iii) the The DNA replication apparatus has been well characterized in existence of an entirely different DNA polymerase in P. Bacteria, with Escherichia coli serving as a model (1, 2). In this furiosus. This uncertainty was clarified when the gene encoding organism, chromosomal duplication is the function of the DP2 in P. furiosus was isolated (17). Arranged in tandem with DNA polymerase III holoenzyme. The genes encoding all 10 this gene, which in actuality codes for a protein with a subunits of the holoenzyme have been identified, and these molecular mass of 143,161 Da, is a smaller gene encoding a proteins have been overproduced, purified, and reconstituted. protein (Pfu DP1) with a molecular mass of 69,294 Da. Nested Proteins with corresponding functions have been identified in deletion analyses of the corresponding genes indicated that Eukarya (3, 4). However, in both Bacteria and Eukarya, few DP1 regulates the level of DNA polymerase activity (17). Some of these similarly functioning proteins exhibit any meaningful biochemical properties of Pol II, such as an excellent primer- amino acid conservation. extension ability (which uses the single-stranded M13 DNA Two decades ago, biologists witnessed a landmark discovery primed with an oligonucleotide) and the strong 39 3 59 by Woese and Fox (5), who announced the existence of a third exonuclease activity (for proofreading), suggest that the DNA form of life, currently referred to as Archaea (6). Even though polymerase is a replicative enzyme (17). members of this domain are dissimilar to the eukaryotes (6), When the total genome sequence of M. jannaschii was archaeal information-processing systems (i.e., transcription, reported (9), we found the homologs of Pfu Pol II and showed translation, and apparently replication systems) are more that these proteins (MJ0702 and MJ1630) have both DNA- similar to the eukaryotic than to the bacterial versions. The polymerizing activity and 39 3 59 exonuclease activity (18). study of archaeal information processing may, therefore, help In this study, we show that polypeptides having sequences us to understand the structure, function, and evolution of similar to Pfu Pol II exist in the genomes of three other homologous eukaryotic systems and vice versa. euryarchaeotes, Methanobacterium thermoautotrophicum (19), Previously, we cloned a DNA-polymerase gene that encodes Archaeoglobus fulgidus (20), and Pyrococcus horikoshii (ref. 21; a an eukaryote-like family B ( -like) DNA polymerase from the for more information see www.bio.nite.go.jp). Furthermore, euryarchaeote Pyrococcus furiosus (7). Furthermore, we the archaeal–eukaryotic relationship is substantiated by the showed that the crenarchaeote Pyrodictium occultum possesses similarity of amino acid sequences of euryarchaeal DP1 and at least two family B DNA polymerases (8). Including our the small subunit of eukaryotic DNA polymerase d (Pol d), a results, every archaeal DNA polymerase sequenced before the protein essential for replication in Eukarya. In DP2 proteins, first complete archaeal genome report of Methanococcus motifs, including invariant carboxylates (Asp) found in the jannaschii (9) was a single-subunit member of family B (10– palm subdomain of nucleotide polymerases, could be pre- 13). dicted. As further evidence that the two proteins constitute a An extremely puzzling observation from the complete ge- heterodimeric DNA polymerase, we show here that DP1 and nome sequence of M. jannaschii was the presence of what is DP2 interact to make up a complex in P. furiosus cells. apparently a single DNA polymerase sequence (9). This We propose that euryarchaeotes have a different type of DNA polymerase from those found in eukaryotic enzymes and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: Pol I, DNA polymerase I; Pol II, DNA polymerase II; accordance with 18 U.S.C. §1734 solely to indicate this fact. Pol d, DNA polymerase d. © 1998 by The National Academy of Sciences 0027-8424y98y9514250-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: ishino@beri. PNAS is available online at www.pnas.org. co.jp. 14250 Downloaded by guest on September 30, 2021 Evolution: Cann et al. Proc. Natl. Acad. Sci. USA 95 (1998) 14251 that the DP2 proteins are likely to be the catalytic subunit of ml of buffer A (50 mM TriszHCl, pH 8.0y2 mM 2-mercapto- this DNA polymerase family. ethanoly1 mM phenylmethylsulfonyl fluoridey10% glycerol). Cells were disrupted by sonication on ice, followed by centrif- 3 MATERIALS AND METHODS ugation for 10 min at 10,000 g. The supernatant was kept on ice until used. All subsequent steps were carried out at room Protein Sequences. The protein sequences used in this study, temperature. Aliquots (30 ml) of protein A-Sepharose (Phar- together with their accession numbers and ORF numbers macia) were washed three times with PBS (10 mM sodium (from archaeal genome projects), are as follows: from P. phosphate, pH 7.5y0.15 M NaCl). The sepharose in each tube furiosus (Pfu DP1 and Pfu DP2: D84670); from M. jannaschii was mixed with one of the following polyclonal antisera that (Mja DP1: F64387, MJ0702) and (Mja DP2: D64503, MJ1630); were raised independently by immunizing rabbits: anti-Pfu Pol from A. fulgidus (Afu DP1: AE000979, AF1790) and (Afu DP2: I (family B DNA polymerase), anti-Pfu DP1, anti-Pfu Pol II AE000984, AF1722); from M. thermoautotrophicum (Mth (DP1 1 DP2), or anti-PI-PfuI [a P. furiosus intein protein DP1: AE000903, MTH1405) and (Mth DP2: AE000913, (K.K., N. Fujita, K. Ichiyanagi, H. Shinagawa, K. Morikawa, MTH1536); from P. horikoshii (Pho DP1: AB009468, and Y.I., unpublished work) as a control]. The mixtures were PHBN023) and (Pho DP2: AB009468, PHBN021); from Homo incubated for1honarotaryshaker, followed by two washes sapiens Pol d small subunit (Hsa PolD: U21090); from Arabi- with PBS and one with buffer A. The sepharose in each tube dopsis thaliana Pol d small subunit (Ath PolD: AC002561); was then mixed with 300 ml of supernatant from the P. furiosus from Caenorhabditis elegans Pol d small subunit (Cel PolD: cell extract and incubated for 30 min on a rotary shaker. Z73425); from Saccharomyces cerevisiae (Sce HYS2: D50324); Western Blotting. The immunoprecipitates, prepared as from Schizosaccharomyces pombe (Spo Cdc1: Y12561); from S. described above, were washed twice with buffer A and mixed cerevisiae transposon TYI protein B (Sce TYI: P47098); from with 240 ml of buffer A and 60 mlof53 sample buffer [0.25 M HIV DNA polymerase polyprotein (HIV RT: P05961); from TriszHCl (pH 6.8)y5% (volyvol) glyceroly5% 2-mercaptoetha- Euplotes aediculatus telomerase subunit (Eau Tel: U95964); noly0.2% bromophenol blue]. The mixture was boiled for 5 from bacteriophage T5 DNA polymerase (T5 Dpol: P19822); min, followed by microcentrifugation at 10,000 3 g for 5 min. from bacteriophage KII DNA-directed RNA polymerase (KII Supernatant (3 ml) was electrophoresed on an SDSy7.5% Rpol: P18147); from Sendai virus RNA polymerase b subunit PAGE, transferred onto a poly(vinylidene difluoride) mem- (Sen Rpol: P06829); from E. coli DNA polymerase I (Pol I; brane (0.2 mm; Bio-Rad) and reacted with each antiserum. The Eco DPolI: P00582); from Thermus aquaticus Pol I (Taq blots were analyzed with the enhanced chemiluminescence DPolI: D32013); from H. sapiens DNA polymerase a (Hsa system (Amersham) incorporated with horseradish peroxi- DPola: P09884); from E. coli Pol II (Eco DPolII: X54847); dase-linked anti-rabbit IgG (Amersham), according to the from S. cerevisiae DNA polymerase « catalytic subunit A (Sce manufacturer’s instructions.
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