Proteolytic cleavage of the hyperthermophilic topoisomerase I from Thermotoga maritima does not impair its enzymatic properties.
Using limited proteolysis, we show that the hyperthermophilic topoisomerase I from Thermotoga maritima exhibits a unique hot spot susceptible to proteolytic attack with a variety of proteases. The remaining of the protein is resistant to further proteolysis, which suggests a compact folding of the thermophilic topoisomerase, when compared to its mesophilic Escherichia coli homologue. We further show that a truncated version of the T. maritima enzyme, lacking the last C-terminal 93 amino acids is more susceptible to proteolysis, which suggests that the C-terminal region of the topoisomerase may be important to maintain the compact folding of the enzyme. The hot spot of cleavage is located around amino acids 326-330 and probably corresponds to an exposed loop of the protein, near the active site tyrosine in charge of DNA cleavage and religation. Location of this protease sensitive region in the vicinity of bound DNA is consistent with the partial protection observed in the presence of different DNA substrates. Unexpectedly, although proteolysis splits the enzyme in two halves, each containing part of the motifs involved in catalysis, trypsin-digested topoisomerase I retains full DNA binding, cleavage, and relaxation activities, full thermostability and also the same hydrodynamic and spectral properties as undigested samples. This supports the idea that the two fragments which are generated by proteolysis remain correctly folded and tightly associated after proteolytic cleavage.
Cossard
Raynald
R
Laboratoire d'Enzymologie des Acides Nucléiques, Institut de Génétique et Microbiologie, UMR 8621 CNRS, Bât. 400, Université de Paris Sud, Centre d'Orsay, 91405 Orsay Cedex, France.
Viard
Thierry
T
Lamour
Valérie
V
Duguet
Michel
M
Bouthier de La Tour
Claire
C
D020816:Amino Acid Motifs; D001426:Bacterial Proteins; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D020124:Thermotoga maritima / Q000201:enzymology*
D001426:Bacterial Proteins; D010446:Peptide Fragments; D004247:DNA; D010450:Endopeptidases; D004264:DNA Topoisomerases, Type I