CLONING AND SEQUENCE-ANALYSIS OF THE DNA LIGASE-ENCODING GENE OF RHODOTHERMUS-MARINUS, AND OVERPRODUCTION, PURIFICATION AND CHARACTERIZATION OF 2 THERMOPHILIC DNA LIGASES

In this paper we describe the cloning and sequence analysis of a gene encoding DNA ligase (Lig; EC 6.5.1.2) from the thermophilic bacterium Rhodothermus marinus (Rm). We also describe the overexpression of the Lig-encoding genes of Rm and the thermophile, Thermus scotoductus (Ts) , in Escherichia coli, and the purification and characterization of th e overproduced Lig. The Rm lig gene encodes a protein of 712 amino aci ds (aa) with a calculated molecular mass of 79 487 Da. Comparison with published sequences of bacterial Lig revealed significant homology be tween the NAD(+)-utilizing Lig, and alignment of their aa sequences re vealed several blocks of conserved residues. Both of the purified Lig exhibit nick-closing activity over a wide range of temperatures. Under our assay conditions the Rm Lig was active at 5-75 degrees C with app arent optimal activity above 55 degrees C. The Ts enzyme showed activi ty at 15-75 degrees C with optimal activity above 65 degrees C. The ha lf-life of the Lig at 91 degrees C was estimated to be 7 min for the R m Lig and 26 min for the Ts Lig.