Abstract : A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine
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Abstract : A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system providing sequence specificity. We identified two genes encoding Vsr endonucleases V.NgoAXIII and V.NgoAXIV from Neisseria gonorrhoeae FA1090 based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli the proteins were biochemically characterized and the endonucleolytic activities and specificities of V.NgoAXIII and V.NgoAXIV were determined. V.NgoAXIII was found to be multispecific and to recognize TG mismatches in every nucleotide context tested whereas V.NgoAXIV recognized TG mismatches in the following sequences GTGG CTGG GTGC ATGC and CTGC. Alanine mutagenesis of conserved residues showed that Asp50 and His68 of V.NgoAXIII and Asp51 and His69 of V.NgoAXIV are essential for hydrolytic activity. Glu25 His64 and Asp97 of V.NgoAXIV and Glu24 Asp63 and Asp97 of V.NgoAXIII are important but not crucial for the activity of V.NgoAXIII and V.NgoAXIV. However Glu24 and Asp63 are also important for the specificity of V.NgoAXIII. On the basis of our results concerning features of Vsr endonucleases expressed by N. gonorrhoeae FA1090 we postulate that at least two types of Vsr endonucleases can be distinguished.
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