Thermotoga maritima-Escherichia coli chimeric topoisomerases. Answers about involvement of the carboxyl-terminal domain in DNA topoisomerase I-mediated catalysis.
Bacterial topoisomerases I are generally composed of two domains as follows: a core domain, which contains all the conserved motifs involved in the trans-esterification reactions, and a carboxyl-terminal domain, highly variable in size and sequence. In the present work, we have addressed the question of the respective roles of the two domains in the different steps of the topoisomerization cycle. For this purpose, we prepared various recombinant topoisomerases from two model enzymes: topoisomerase I from the hyperthermophilic bacterium Thermotoga maritima and topoisomerase I from Escherichia coli. We compared the properties of the two core domains to that of the topoisomerases formed by combining the core domain of one enzyme to the carboxyl-terminal domain of the other. We found that, contrary to E. coli (Lima, C. D., Wang, J. C., and Mondragon, A. (1993) J. Mol. Biol. 232, 1213-1216), the core domain from T. maritima (TmTop65) is able to sustain by itself a complete topoisomerization cycle, although with low efficiency. Fusion of TmTop65 to the entire carboxyl-terminal domain from E. coli considerably increases binding efficiency, thermal stability, and DNA relaxation activity. Moreover, the chimera predominantly acquires the cleavage specificity of E. coli full-length topoisomerase. For the chimera obtained by fusion of the T. maritima carboxyl-terminal domain to the core EcTop67, very low DNA relaxation activity and binding are recovered, but formation of a covalent DNA adduct is impaired. Taken together, our results show that the presence and the nature of the carboxyl-terminal domain of bacterial topoisomerases I strongly determine their DNA binding efficiency and cleavage specificity but is not strictly required for strand passage.
Viard
Thierry
T
Laboratoire d'Enzymologie des Acides Nucléiques, Institut de Génétique et Microbiologie, UMR 8621 CNRS, Bātiment 400, Université Paris Sud, Centre d'Orsay, 91405 Orsay, France.
Cossard
Raynald
R
Duguet
Michel
M
de La Tour
Claire Bouthier
CB
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D018388:Codon, Terminator; D004247:DNA / Q000737:chemistry; D018736:DNA Adducts; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology*; D013997:Time Factors; D015032:Zinc / Q000737:chemistry
D018388:Codon, Terminator; D018736:DNA Adducts; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA; D004264:DNA Topoisomerases, Type I; D015032:Zinc