A DNA Polymerase III Holoenzyme-like Subassembly from an Extreme Thermophilic Eubacterium Charles S. McHenry 1 *, Mark Seville 2 and Millard G. Cull 2 1 Department of Biochemistry and Molecular Genetics and the Molecular Biology Program University of Colorado Health Sciences Center, Denver CO 80262, USA 2 Enzyco, Inc., 191 University Blvd., Ste. 305, Denver CO 80206, USA We have puri®ed a novel DNA polymerase from Thermus thermophilus. This was enabled by use of general gap ®lling assays to monitor poly- merase activity and cross-reactive monoclonal antibodies against the a catalytic subunit of E. coli DNA polymerase III holoenzyme to distinguish a novel polymerase from the well characterized DNA polymerase I-like Thermus thermophilus DNA polymerase. Two proteins migrating with the polymerase after three chromatographic steps were isolated and sub- jected to partial amino acid sequencing. The amino termini of both were homologous to the two products of the E. coli dnaX gene, the g and t sub- units of the DNA polymerase III holoenzyme. Using this information and sequences conserved among dnaX-like genes, we isolated a gene fragment by PCR and used it as a probe to isolate the full length Thermus thermo- philus dnaX gene. The deduced amino acid sequence is highly homolo- gous to the DnaX proteins of other bacteria. Examination of the sequence permitted identi®cation of a frameshift site similar to the one used in E. coli to direct the synthesis of the shorter g DnaX-gene product. Based on this information, we conclude that a conventional replicase exists in extreme thermophilic eubacteria. The general biological and practical technological implications of this ®nding are discussed. # 1997 Academic Press Limited Keywords: thermophile; T. thermophilus; DNA polymerase; holoenzyme; DNA replication *Corresponding author Introduction Escherichia coli, an often studied prototype for general eubacterial mechanisms, encodes three dis- tinct DNA polymerases. DNA polymerase I is the most abundant polymerase and is responsible for some types of DNA repair, including a repair-like reaction that permits the joining of Okazaki frag- ments during DNA replication. DNA polymerase III comprises the catalytic core of the E. coli repli- case. There are approximately 400 copies of DNA polymerase I per cell, but only 10 to 20 copies of Pol III (Kornberg & Baker, 1992; Wu et al., 1984). The low abundance of Pol III and its relatively fee- ble activity on gapped DNA templates typically used as a general replication assay delayed its dis- covery until the availability of mutants defective in DNA polymerase I (Kornberg & Gefter, 1972). The catalytic subunit of Pol III is distinguished from other polymerases as a component of E. coli major replicative complex, apparently not by its intrinsic catalytic activity, but by its ability to inter- act with other replication proteins at the fork. These interactions confer upon the enzyme enor- mous processivity. Once the DNA polymerase III holoenzyme associates with primed DNA, it does not dissociate for over 40 minutes, the time required for the synthesis of the entire 4 Mb E. coli chromosome (McHenry, 1988). Studies in coupled rolling circle models of the replication fork suggest the enzyme can synthesize DNA 150 kb or longer without dissociation in vitro (Mok & Marians, 1987; Wu et al., 1992). The essential interaction required for this high processivity is an interaction between the a catalytic subunit and a dimer of b, a sliding clamp processivity factor that encircles the DNA template like a bracelet, permitting it to rapidly slide along with the associated polymerase, but preventing it from falling off (LaDuca et al., 1986; Kong et al., 1992). The b 2 bracelet cannot spontaneously associate with high molecular mass DNA, it requires a mul- tiprotein DnaX-complex to open and close it around DNA using the energy of ATP hydrolysis (Wickner, 1976; Naktinis et al., 1995; Dallmann Abbreviations used: Pol, polymerase; Fr, fraction. J. Mol. Biol. (1997) 272, 178±189 0022±2836/97/370178±12 $25.00/0/mb971238 # 1997 Academic Press Limited