Site-Directed Mutagenesis of the T4 Endonuclease V Gene: Role of Lysine-130

Adrian Recinos, R. Stephen Lloyd

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Abstract

The DNA sequence of the bacteriophage T4 denV gene which encodes the DNA repair enzyme endonuclease V was previously constructed behind the hybrid X promoter OLPRin a plasmid vector. Theolpr-denV sequence was subcloned in M13mpl8 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 > H-130 > G-130 » control denV-. A surprising exception to this trend was that G-130, while low in dimer-specific binding capacity, demonstrated an AP-endonuclease function several times as efficient as that of the wild-type enzyme. Overall, these results suggest that the alterations of Lys-130 chiefly compromise the ability of the mutant enzymes to bind DNA at dimer sites. Dimer-specific binding, however, appears not to be required for the AP-endonuclease activity.

Original languageEnglish (US)
Pages (from-to)1832-1838
Number of pages7
JournalBiochemistry
Volume27
Issue number6
DOIs
StatePublished - Mar 1 1988

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ASJC Scopus subject areas

  • Biochemistry

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