Cloning of a gene from Thermus filiformis and characterization of the thermostable nuclease

Abstract

A gene coding for a thermostable nuclease was cloned from the thermophilic microorganism, Thermus filiformis (Tf), using an indicator strain containing a dinD::lacZ fusion. The gene, designated nuc17, has been mapped within a 2300-bp fragment. The 55-kDa Tf nuclease was purified to over 95% homogeneity. Single-stranded (ss) DNA is the preferred substrate for the Tf nuclease, although double-stranded (ds) DNA can also be digested. Nuclease activity increases with increasing temperature up to 80 degrees C and requires the metal ions Ca++ or Mg++ for catalysis. Tf nuclease is primarily an endonuclease that leaves 5' phosphates in the digested products. The ssDNA extensions remaining after exonuclease III digestion of dsDNA can be removed by the Tf nuclease, making it a useful reagent to generate unidirectional deletions.