Characterization of intein homing endonuclease encoded in the DNA polymerase gene of Thermococcus marinus

Heejin Bae, Kee Pum Kim, Jung Min Song, Jun Hwan Kim, Joo Sung Yang, Suk Tae Kwon

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)180-188
Number of pages9
JournalFEMS Microbiology Letters
Volume297
Issue number2
DOIs
StatePublished - Aug 2009

Keywords

  • Endonuclease
  • Intein
  • Thermococcus marinus

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