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. 2000 Nov 1;28(21):4391-6.
doi: 10.1093/nar/28.21.4391.

The thy pol-2 intein of Thermococcus hydrothermalis is an isoschizomer of PI-TliI and PI-TfuII endonucleases

I Saves  1 , H Eleaume, J Dietrich, J M Masson
Affiliations

The thy pol-2 intein of Thermococcus hydrothermalis is an isoschizomer of PI-TliI and PI-TfuII endonucleases

I Saves et al. Nucleic Acids Res. .

Abstract

THY Pol-2 intein, from Thermococcus hydrothermalis, belongs to the same allelic family as TLI Pol-2 (PI-TLII), Tfu Pol-2 (PI-TFUII) and TspTY Pol-3 mini-intein, all inserted at the pol-c site of archaeal DNA polymerase genes. This new intein was cloned, expressed in Escherichia coli and purified. The intein is a specific endonuclease (PI-THY:I) which cleaves the inteinless sequence of the THY DNA pol gene. Moreover, PI-TLII, PI-TFUII and PI-THYI are very similar endonucleases which cleave DNA in the same optimal conditions at 70 degrees C yielding similar 3'-hydroxyl overhangs of 4 bp and the reaction is subject to product inhibition. The three enzymes are able to cleave the three DNA sequences spanning the pol-c site and a 24 bp consensus cleavage site was defined for the three isoschizomers. However, the exact size of the minimal cleavage site depends both on the substrate sequence and the endonuclease. The inability of the isoschizomers to cleave the inteinless DNA polymerase gene from Pyrococcus spp. KOD is due to point substitutions on the 5' side of the pol-c site, suggesting that the absence of inteins of this allelic family in DNA polymerase genes from Pyrococcus spp. can be linked to small differences in the target site sequence.

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Figures

Figure 1
Figure 1
Inteins in DNA polymerases of archaebacteria. The three families of allelic inteins are presented. Names of inteins, lengths in amino acids of each extein and intein and GenBank accession numbers of DNA polymerases are indicated. Names for the endonuclease activities, which have been experimentally demonstrated, are indicated in parentheses after the intein names. Tfu DNA Pol, Tsp TY DNA Pol, Tli DNA Pol, Thy DNA Pol, Psp KOD DNA Pol, Mja DNA Pol, Psp GBD DNA Pol and Pho DNA Pol are the abreviations for the DNA polymerases from Thermococcus fumicolans, Thermococcus spp. TY, Thermococcus litoralis, T.hydrothermalis, Pyrococcus spp. KOD, M.jannaschii, Pyrococcus spp. GBD and Pyrococcus horikosshi OT3, respectively.
Figure 2
Figure 2
(A) Cleavage assay for PI-ThyI. Linearised (100 ng) Thy-site substrate were incubated either with (+) or without (–) 2 ng of PI-ThyI for 10 min at 70°C in a 50 mM Tris–acetate pH 8 buffer containing 75 mM Mg(OAc)2 and 100 mM NH4OAc. Substrate (S; 2730 bp) and products (P; 940 and 1790 bp) were separated on a 1% agarose gel in TBE buffer. (B) Minimal recognition sequence of PI-ThyI. Minimal nucleotide sequence necessary for recognition and cleavage by PI-ThyI was determined by primer extension on the plasmid Thy-site. The dotted line indicates the homing site of the intein in the DNA Pol gene. The dashed box delimitates the minimal recognition site and arrows designate the cleavage points on each DNA strand.
Figure 3
Figure 3
Minimal recognition sequence of PI-ThyI, PI-TfuII and PI-TliI on each pol-c site. The sequences of pol-c intein insertion sites in Thy, Tfu and Tli DNA pol genes are indicated. Minimal recognition and cleavage sequences of these three DNA sequences by PI-ThyI, PI-TfuII and PI-TliI appear in red, green and blue, respectively. Nucleotides that are not conserved in the three sequences appear in bold. The 4 bp overhangs generated by the cleavage reactions are underlined.
Figure 4
Figure 4
Minimal cleavage sites of five different substrates by PI-ThyI. The large black lines under the five sequences indicate the minimal sequences necessary for cleavage. Nucleotides that are different from the wild-type PI-ThyI site appear in bold.
Figure 5
Figure 5
Specific activities of PI-TfuII on four substrates, expressed in 104 U/mg. Various DNA substrates are symbolised by black or white boxes representing Psp KOD and Tfu DNA Pol gene sequences, respectively.
Figure 6
Figure 6
Sequences at the pol-c site in eight DNA Pol genes from archaebacteria. The 24 bp sequences spanning the pol-c site in DNA pol, with or without an intein at this site, are aligned with the consensus cleavage site. Nucleotides that diverge from this consensus appear in bold.
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