Nucleotide sequence of the gene for a 74 kDa DNA polymerase from the archaeon Sulfolobus solfataricus.

Recently, the nucleotide sequence of a DNA polymerase gene from Sulfolobus solfataricus was reported (1). The enzyme is a 100 kDa polypeptide also possessing a 3'—5' exonuclease activity. In addition to this enzyme in cells of Sulfolobus we have detected the presence of a smaller DNA polymerase devoid of any associated nuclease activities (2). We made an attempt to clone and sequence a gene for the latter enzyme. An oligonucleotide 5' -GAGAT(A/T)G AGGG(G/T)GA(G/T) ATGGG-3' which corresponds to the conserved region immediately upstream of the sequence motif YGDTD almost invariant in family B DNA polymerases (3) was used to probe with low-stringency hybridization Southern blots of S. solfataricus P2 DNA digested with a variety of restriction enzymes. For each digest a single fragment was detected hybridizing with the probe used. We cloned the 5.7 kbp BamHI—Clal fragment into pBluescript II and sequenced both strands of it by the dideoxynucleotide chain termination method. An 1878 bp long open reading frame, corresponding to a protein of a molecular mass of 73.532 Da, was found within the sequenced region. The open reading frame is in a transcribed region: the in vivo transcript start was mapped by primer extension analysis using the oligonucleotide 5'-TCACTCCTCCCTTGATTGG-3' as a primer (data not shown). Twenty six nucleotides upstream of the transcript start a sequence corresponding to a standard archaeal boxA (4) was detected. Downstream of the gene a typical archaeal terminator sequence (5) is present.