Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of two 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 the overproduced Lig. The Rm lig gene encodes a protein of 712 amino acids (aa) with a calculated molecular mass of 79,487 Da. Comparison with published sequences of bacterial Lig revealed significant homology between the NAD(+)-utilizing Lig, and alignment of their aa sequences revealed 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 apparent optimal activity above 55 degrees C. The Ts enzyme showed activity at 15-75 degrees C with optimal activity above 65 degrees C. The half-life of the Lig at 91 degrees C was estimated to be 7 min for the Rm Lig and 26 min for the Ts Lig.
Thorbjarnardóttir
S H
SH
Institute of Biology, University of Iceland, Reykjavik.
Jónsson
Z O
ZO
Andrésson
O S
OS
Kristjánsson
J K
JK
Eggertsson
G
G
Palsdottir
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D011088:DNA Ligases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D007527:Isoenzymes; D011088:DNA Ligases