Identification of nucleases and phosphatases by direct biochemical screen of the Saccharomyces cerevisiae proteome.
The availability of yeast strain collections expressing individually tagged proteins to facilitate one-step purification provides a powerful approach to identify proteins with particular biochemical activities. To identify novel exo- and endo-nucleases that might function in DNA repair, we undertook a proteomic screen making use of the movable ORF (MORF) library of yeast expression plasmids. This library consists of 5,854 yeast strains each expressing a unique yeast ORF fused to a tripartite tag consisting of His(6), an HA epitope, a protease 3C cleavage site, and the IgG-binding domain (ZZ) from protein A, under the control of the GAL1 promoter for inducible expression. Pools of proteins were partially purified on IgG sepharose and tested for nuclease activity using three different radiolabeled DNA substrates. Several known nucleases and phosphatases were identified, as well as two new members of the histidine phosphatase superfamily, which includes phosphoglycerate mutases and phosphatases. Subsequent characterization revealed YDR051c/Det1 to be an acid phosphatase with broad substrate specificity, whereas YOR283w has a broad pH range and hydrolyzes hydrophilic phosphorylated substrates. Although no new nuclease activities were identified from this screen, we did find phosphatase activity associated with a protein of unknown function, YOR283w, and with the recently characterized protein Det1. This knowledge should guide further genetic and biochemical characterization of these proteins.
Ho
Chu Kwen
CK
Department of Microbiology, Columbia University College of Physicians & Surgeons, New York, New York, USA.
Lam
Alicia F
AF
Symington
Lorraine S
LS
D004260:DNA Repair; D003851:Deoxyribonucleases / Q000737:chemistry*; D000939:Epitopes / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008957:Models, Genetic; D016366:Open Reading Frames; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D020543:Proteome; D040901:Proteomics / Q000379:methods; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D000939:Epitopes; D005656:Fungal Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D003851:Deoxyribonucleases; D010744:Phosphoric Monoester Hydrolases