Site-directed mutagenesis of the T4 endonuclease V gene: role of lysine-130

• Published in: Biochemistry (Easton) 1988-03, Vol.27 (6), p.1832-1838
• Main Authors: Recinos, Adrian, Lloyd, R. Stephen
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Deoxyribonuclease (Pyrimidine Dimer); Endodeoxyribonucleases - genetics; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - radiation effects; Fundamental and applied biological sciences. Psychology; Genes; Genes, Viral; Genes. Genome; Kinetics; Lysine; Molecular and cellular biology; Molecular genetics; Mutation; phage T4; Plasmids; Promoter Regions, Genetic; T-Phages - enzymology; T-Phages - genetics; Ultraviolet Rays; Viral Proteins
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00406a006

The DNA sequence of the bacteriophage T4 denV gene which encodes the DNA repair enzyme endonuclease V was previously constructed behind the hybrid lambda promoter OLPR in a plasmid vector. The OLPR-denV sequence was subcloned in M13mp18 and used as template to construct site-specific mutations in the denV structural gene in order to investigate structure/function relationships between the primary structure of the protein and its various DNA binding and catalytic activities. The Lys-130 residue of the wild-type endonuclease V has been postulated to be associated with its apurinic endonuclease (AP-endonuclease) activity. The codon for Lys-130 was changed to His-130 or Gly-130, and each denV sequence was subcloned into a pEMBL expression vector. These plasmids were transformed into repair-deficient Escherichia coli (uvrA recA), and the following parameters were examined for cells or cell extracts: expression and accumulation of endonuclease V protein (K-130, H-130, or G-130); survival after UV irradiation; dimer-specific DNA binding; and kinetics of phosphodiester bond scission at pyrimidine dimer sites, dimer-specific N-glycosylase activity, and AP-endonuclease activity. The enzyme's intracellular accumulation was significantly decreased for G-130 and slightly decreased for H-130 despite normal levels of denV-specific mRNA for each mutant. On a molar basis, the endonuclease V gene products generally gave parallel levels of each of the catalytic and binding functions with K-130 greater than H-130 greater than G-130 much greater than control denV-.