The experimental platform can be readily extended to additional humanized yeast models of human disease genes and therapeutic targets, adding systematic generation of putative trapping alleles by deep mutational scanning. that encodes a putative trapping version of FEN1, in the presence AR234960 of the normal gene. Systematic genetic interaction screening results in a genetic interaction profile for the trapping allele of (6) and (7), have expanded to systematic studies to identify the complete set of human genes that can replace their yeast orthologs to create what are termed humanized yeast (8). The resulting humanized yeast can then be used in a myriad of assays to analyze the function of the human gene, most notably in screening small molecules for activity against human protein targets, including PARP inhibitors (9, 10). Interestingly, even in cases where an entire human gene does not complement deletion of its yeast ortholog, humanization of regions or specific amino acids of the yeast gene can be fruitful, particularly in modeling human disease-relevant mutations (11, 12). Also, if expression of a human gene elicits a growth phenotype in yeast, genetic screens to enhance or suppress the phenotype can reveal new disease-relevant biology [e.g., alpha-synuclein (13)]. The study by Hamza et al. (5) offers AR234960 an interesting twist on humanized yeast. The authors show that a human gene, in this case alleles that were designed to inactivate catalysis but not DNA binding, and so were likely to result in trapping of FEN1CDNA complexes. Deep mutational scanning could be used to make separation of function mutants that lose catalytic activity but that retain proteinCDNA or proteinCprotein interactions (15), exactly the type of trapping mutants that Hamza et al. model with their alleles. Systematic Screening of Specialized Alleles The effort AR234960 of Hamza et al. (5) to discover genetic interactions associated with expression of a specific allele of complements and leverages major efforts in the yeast community to systematically map genetic interactions. The budding yeast is the only eukaryotic system for which a complete map of synthetic lethal interactions has been generated, through tests of all possible double mutants for synthetic growth defects (16, 17). Genetic networks can be mapped on this scale using methods for automated yeast genetics, which enable rapid generation of double mutants (17), and arrayed collections of yeast mutants carrying deletion alleles of nonessential genes or hypomorphic alleles of essential genes (for example, temperature-sensitive alleles) (18). Analysis of genetic interaction profiles, or the set of genes that show genetic interactions with a particular query strain, provides functionally rich information and identifies genes that share roles in various biological pathways and processes (17). The genetic interaction profile associated with deletion of (5). However, the dominant mutant allele of is only synthetic lethal with homologous recombination mutants, thus revealing a specific genetic interaction profile that a desirable trapping drug should match (Fig. 1are synthetic lethal with homologous recombination deficiencies. They predict that humanized yeast expressing in a recombination deficient background should be synthetic lethal with FEN1 inhibitors that cause trapping of FEN1CDNA complexes. The experimental platform can be readily extended to additional humanized yeast models of human disease genes and therapeutic targets, adding systematic generation of putative trapping alleles by deep mutational scanning. Panels of mutants can then be fed into specialized genetic interaction screens with increasingly sophisticated readouts, including fitness, morphology, protein abundance, and protein localization (23). The detailed genetic interaction profiles that result can form the basis for small molecule screens carefully honed to identify lead compounds with very specific properties. Acknowledgments Work in the laboratories of G.W.B. and B.A. is supported by grants from the Canadian Institutes of Health Research (FDN-159913 to G.W.B.; FDN-143265 to B.A.), the Canadian Cancer Society (706293 to G.W.B.), the Natural Sciences and Engineering Research Council of Canada (RGPIN-2017-06855 to G.W.B.), and the National Institutes of Health (R01HG005853 to B.A.). Footnotes The authors declare no competing interest. See companion article, Modeling DNA trapping of anticancer therapeutic targets using missense mutations identifies dominant synthetic lethal interactions, 10.1073/pnas.2100240118..is supported by grants from the Canadian Institutes of Health Research (FDN-159913 to G.W.B.; FDN-143265 to B.A.), the Canadian Cancer Society (706293 to G.W.B.), the Natural Sciences and Engineering Research Council of Canada (RGPIN-2017-06855 to G.W.B.), and the National Institutes of Health (R01HG005853 to B.A.). Footnotes The authors declare no competing interest. See companion article, Modeling DNA trapping of anticancer therapeutic targets using missense mutations identifies dominant synthetic lethal interactions, 10.1073/pnas.2100240118.. PARP inhibitors (9, 10). Interestingly, even in cases where an entire human gene does not complement deletion of its yeast ortholog, humanization of regions or specific amino acids of the yeast gene can be fruitful, particularly in modeling human disease-relevant mutations (11, 12). Also, if expression of a human gene elicits a growth phenotype in yeast, genetic screens to enhance or suppress the phenotype can reveal new disease-relevant biology [e.g., alpha-synuclein (13)]. The study by Hamza et al. (5) offers an interesting twist on humanized yeast. The authors show that a human gene, in this case alleles that were designed to inactivate catalysis but not DNA binding, and so were likely to result in trapping of FEN1CDNA complexes. Deep mutational scanning could be used to make separation of function mutants that lose catalytic activity but that retain proteinCDNA or proteinCprotein interactions (15), exactly the type of trapping mutants that Hamza et al. model with their alleles. Systematic Screening of Specialized Alleles The effort of Hamza et al. (5) to discover genetic interactions associated with expression of a specific allele of complements and leverages major efforts in the yeast community to systematically map genetic interactions. The budding yeast is the only eukaryotic system for which a complete map of synthetic lethal interactions has been generated, through tests of all possible double mutants MGC18216 for synthetic growth defects (16, 17). Genetic networks can be mapped on this scale using methods for automated yeast genetics, which enable rapid generation of double mutants (17), and arrayed collections of yeast mutants carrying deletion alleles of nonessential genes or hypomorphic alleles of essential genes (for example, temperature-sensitive alleles) (18). Analysis of genetic interaction profiles, or the set of genes that show genetic interactions with a particular query strain, provides functionally rich information and identifies genes that share roles in various biological pathways and processes (17). The genetic interaction profile associated with deletion of (5). However, the dominant mutant allele of is only synthetic lethal with homologous recombination mutants, thus revealing a specific genetic interaction profile that a desirable trapping drug should match (Fig. 1are synthetic lethal with homologous recombination deficiencies. They predict that humanized yeast expressing in a recombination deficient background should be synthetic lethal with FEN1 inhibitors that cause trapping of FEN1CDNA complexes. The experimental platform can be readily extended to AR234960 additional humanized yeast models of human disease genes and therapeutic targets, adding systematic generation of putative trapping alleles by deep mutational scanning. Panels of mutants can then be fed into specialized genetic interaction screens with increasingly sophisticated readouts, including fitness, morphology, protein abundance, and protein localization (23). The detailed genetic interaction profiles that result can form the basis for small molecule screens carefully honed to identify lead compounds with very specific properties. Acknowledgments Work in the laboratories of G.W.B. and B.A. is supported by grants from the Canadian Institutes of Health Research (FDN-159913 to G.W.B.; FDN-143265 to B.A.), the Canadian Cancer Society (706293 to G.W.B.), the Natural Sciences and Engineering Research Council of Canada (RGPIN-2017-06855 to G.W.B.), and the National Institutes of Health (R01HG005853 to B.A.). Footnotes The authors declare no competing interest. See companion article, Modeling DNA trapping of anticancer therapeutic targets using missense mutations identifies dominant synthetic lethal interactions, 10.1073/pnas.2100240118..

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