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Analysis of the Role of the Cysteine 171 Residue in the Activity of Herpes Simplex Virus Type 1 Thymidine Kinase by Oligonucleotide-directed Mutagenesis

Graham Darby

Division of Virology, Department of Pathology, Cambridge University, Tennis Court Road, Cambridge CB2 1QP, U.K.

The thymidine kinase (TK) gene from herpes simplex virus type 1 strain SC16 was cloned into bacteriophage M13 mp8 so that functional HSV-1 TK was expressed in bacteria infected with the recombinant bacteriophage, M13/TK. Oligonucleotide site-directed mutagenesis was then employed to introduce single nucleotide changes into the TK gene in M13/TK in order to alter the codon for cysteine 171 in the wild-type enzyme to a codon specifying either serine or glycine. Analysis of the mutant enzymes in bacterial extracts showed that these substitutions had little effect on the activity of the enzyme, indicating that the side chain of this residue is not involved in nucleoside binding and is not essential for the catalytic activity of the enzyme.

Present address: Department of Biochemical Virology, Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, U.K.

Received 11 June 1986; accepted 19 September 1986.