Site-directed mutagenesis of the T4 endonuclease V gene: role of tyrosine-129 and -131 in pyrimidine dimer-specific binding

Stump, Donald G.; Lloyd, R. Stephen

doi:10.1021/bi00406a007

Cited by 33 publications

(15 citation statements)

References 22 publications

(28 reference statements)

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“…Furthermore, site-directed mutagenesis of this region has suggested the importance of aromatic residues in binding to the pyrimidine dimer (6,7). In terms ofthe processive binding ability of T4 endo V to DNA, proteins bearing mutations ofArg-3, -26, or -33 to glutamine were shown to have decreased affinity for nontarget DNA (8)(9)(10).…”

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confidence: 99%

Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V.

Doi

Recktenwald

Karaki

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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T4 endonuclease V [endodeoxyribonucease (pyrimidine dimer); deoxyribonuclease (pyrimikine dimer), EC 3.1.25.1] initiates repafr of damaed DNA by hydrolysis of the N-glycosyl bond at the 5' side of a pyrimidine photodimer in double-stranded DNA. To study one of the active sites of T4 endonudease V, systematic site-directed mutagenesis was performed on the synthetic T4 endonuclease V gene, in paraflel with three-dimensional structure analysis by x-ray crystallography. The mutant proteins were evaluated for DNA glycosylase activity using an oligonucleotide duplex (14-mer) containing a single thymidine dimer as a substrate. Replacement of either Glu-23 with glutamine or asparatic acid or Arg-3 with glutami completely abolished DNA glycosylase activity. Mutation of Arg-3 to lysine or of Arg-26 to glu or lysine in a basic amino acid cluster caused serious defects in DNA glycosylase activity, which are reflected in the increases in K. and decreases in kt of DNA glycosylase activity. On the other hand, substitutions of lysine for Arg-22 or of glutamine for Arg-117 or Lys-121 resulted in increases in the Km value. The completely inactive mutant proteins, E23Q and R3Q, in which glutamie was substituted for Glu-23 and Arg-3, respectively, were further investigated by CD spectroscopy for their ability to bind the oligonucleotide substrate. It was found that the E23Q protein retained specific substrate-binding ability, whereas the R3Q protein did not. These results indicate that Glu-23 plays an important role in catalysis of the DNA glycosylase reaction, and that Arg-3 is a crucial residue for substrate binding. In addition, Arg-22, Arg-26, Arg-117, and Lys-121 in the basic amino acid cluster also participate in substrate binding. We conclude that the basic amino acid cluster in T4 endonuclease V is an essential structure for DNA glycosylase activity.Endonuclease V [endo V; endodeoxyribonuclease (pyrimidine dimer); deoxyribonuclease (pyrimidine dimer), EC 3.1.25.1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a pyrimidine dimer DNA glycosylase (PD glycosylase) and an apyrimidinic/apurinic (AP) endonuclease ( Fig. 1) (1, 2). The gene (T4 denV) encoding T4 endo V was identified in association with the UV resistance of T4 phage and was shown to encode 138 amino acids (Fig. 2) (3). The protein recognizes a pyrimidine photodimer structure in a double-stranded DNA, hydrolyzes the N-glycosyl bond at the 5' side of a pyrimidine dimer, and cleaves the apyrimidinic phosphodiester bond via a (3-elimination reaction. In addition, T4 endo V is capable of binding and scanning nontarget DNA processively (4). The fact that such a small protein possesses two catalytic activities has engendered considerable interest in terms of the relationship between the two activities and the domain structure. As yet, no catalytic mechanism of any PD glycosylase has been elucidated.The aromatic residue-rich region, Trp-Tyr-Lys-Tyr-Tyr (residues 128-132), near the C terminus of ...

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confidence: 99%

Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V.

Doi

Recktenwald

Karaki

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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“…The gene of T4 phage deriV has been cloned (Lloyd & Hanawalt, 1981; Valerie et al, 1984;Radany et al, 1984), and the sequence of the 138 amino acids of this endonuclease was deduced from the base sequence (Valerie et al, 1984). The cloned gene has been expressed in Escherichia coli, and site-directed mutagenesis of the gene has been reported (Recinos & Lloyd, 1988; Stump & Lloyd, 1988). In contrast to these approaches, we have previously synthesized a gene for this endonuclease by ligation of oligonucleotide fragments; this gene was expressed efficiently in E. coli under the control of the trp promoter (Inaoka et al, 1989).…”

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In vitro and in vivo activities of T4 endonuclease V mutants altered in the C-terminal aromatic region

Ishida

Kanamori²,

Hori³

et al. 1990

Biochemistry

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Genes encoding mutants of the thymine photodimer repair enzyme from bacteriophage T4 (T4 endonuclease V) having an amino acid substitution (T127M, W128A, W128S, Y129A, K130L, Y131A, Y132A) were constructed by use of a previously obtained synthetic gene and expressed in Escherichia coli under the control of the E. coli tryptophan promoter. An in vitro assay of partially fractionated mutant proteins for glycosylase activity was performed with chemically synthesized substrates containing a thymine photodimer. T127M and K130L showed almost the same activity as the wild-type protein. Although W128S, Y131A, and Y132A were slightly active, W128A and Y129A lost activity. The results indicated that the aromatic amino acids around position 130 may be important for the glycosylase activity. Mutant T127M was purified, and the Km value was found to be of the same order as that of the wild type (10(-8) M). In vivo activities for all mutants were characterized with UV-sensitive E. coli. The results showed that substitution of Thr-127 with Met or Lys-130 with Leu did not have an effect on the survival of the bacteria but substitution of aromatic amino acids (128-132) had various effects on survival.

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“…Both peptides were degraded by <50% after 1 day. By comparison, the decapeptide XP (WFKYYGKAIY, with free termini), 60 which we used as a positive control, was totally degraded in 3 h ( Figure S4 ). Similarly, OP was stable in DMEM for 3 days (data not shown).…”

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confidence: 99%

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein–Protein Interactions

et al. 2021

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We developed a new class of inhibitors of protein–protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2–20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein–protein interactions in the function of SHP2.

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Site-directed mutagenesis of the T4 endonuclease V gene: role of tyrosine-129 and -131 in pyrimidine dimer-specific binding

Cited by 33 publications

References 22 publications

Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V.

Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V.

In vitro and in vivo activities of T4 endonuclease V mutants altered in the C-terminal aromatic region

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein–Protein Interactions

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