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The DNA polymerase gene of Thermococcus marinus ( Tma) contains an intein inserted at the pol-b site that possesses a 1611-bp ORF encoding a 537-amino acid residue. The LAGLIDADG motif, often found in site-specific DNA endonucleases, was detected within the amino acid sequence of the intein. The intein endonuclease, denoted as PI- Tma, was purified as a naturally spliced product from the expression of the complete DNA polymerase gene in Escherichia coli. PI- Tma cleaved intein-less DNA sequences, leaving four-base-long, 3′-hydroxyl overhangs with 5′-phosphate. Nonpalindromic recognition sequences 19 bp long were also identified using partially complementary oligonucleotide pair sequences inserted into the plasmid pET-22b(+). Cleavage by PI- Tma was optimal when present in 50 mM glycine–NaOH (pH 10.5), 150 mM KCl and 12 mM MgCl2 at 70 °C. [ABSTRACT FROM AUTHOR]