Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans.

Abstract:

Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-->5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-->5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.

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