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J. Biol. Chem., Vol. 278, Issue 11, 9733-9739, March 14, 2003

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Amino Acid Substitutions at Position 43 of NaeI Endonuclease
EVIDENCE FOR CHANGES IN NaeI STRUCTURE^*

Kevin L. Carrick and Michael D. Topal

From the Lineberger Comprehensive Cancer Center and the Department of Biochemistry & Biophysics, University of North Carolina Medical School, Chapel Hill, North Carolina 27599-7295

NaeI endonuclease contains a 10-amino acid region with sequence similarity to the active site KXDG motif of DNA ligase except for leucine (Leu-43) in NaeI (⁴³LXDG⁴⁶). Changing Leu-43 to lysine abolishes the NaeI endonuclease activity and replaces it with topoisomerase and recombinase activities. Here we report the results of substituting Leu-43 with alanine, arginine, asparagine, glutamate, and histidine. Quantitating specific activities and DNA binding values for the mutant proteins determined the range of amino acids at position 43 that alter NaeI mechanism. Substituting alanine, asparagine, glutamate, and histidine for Leu-43 maintained endonuclease activity, but at a lower level. On the other hand, substituting positively charged arginine, like lysine at position 43, converted NaeI to a topoisomerase with no observable double-strand cleavage activity. The specific activities of NaeI-43K and NaeI-43R and their relative sensitivities to salt, the topoisomerase-inhibiting drug N-[4-(9-acridinylamino)-3-methoxyphenyl]methane-sulfonamide (amsacrine) and single-stranded DNA showed that the two activities are similar. The effect of placing a positive charge at position 43 on NaeI structure was determined by measuring (for NaeI and NaeI-43K) relative susceptibilities to proteolysis, UV, circular dichroism spectra, and temperature melting transitions. The results provide evidence that a positive charge at position 43 induces dramatic changes in NaeI structure that affect both the Endo and Topo domains of NaeI. The identification of four putative DNA ligase motifs in NaeI leads us to speculate that structural changes that superimpose these motifs on the ligase structure may account for the changes in activity.

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