Moloney murine leukemia virus (M-MuLV) integrase (IN) catalyses the insertion of the viral genome into the host chromosomal DNA. The limited solubility of the recombinant protein produced in Escherichia coli led the authors to explore the use of Saccharomyces cerevisiae for expression of M-MuLV IN. IN was expressed in yeast and purified by chromatography on nickel-NTA agarose. IN migrated as a single band in SDS-PAGE and did not contain IN degradation products. The enzyme was about twofold more active than the enzyme purified from E. coli and was free of nucleases. Using the yeast system, the substitution of the putative catalytic amino acid Asp184 by alanine was also analysed. The mutated enzyme was inactive in the in vitro assays. This is the first direct demonstration that mutation of Asp184 inactivates M-MuLV IN. Finally, S. cerevisiae was used as a model to assess the ability of M-MuLV IN to interact with eukaryotic protein partners. The expression of an active M-MuLV IN in yeast strains deficient in RAD52 induced a lethal effect. This phenotype could be attributed to cellular damage, as suggested by the viability of cells expressing inactive D184A IN. Furthermore, when active IN was expressed in a yeast strain lacking the ySNF5 transcription factor, the lethal effect was abolished, suggesting the involvement of ySNF5 in the cellular damage induced by IN. These results indicate that S. cerevisiae could be a useful model to study the interaction of IN with cellular components in order to identify potential counterparts of the natural host.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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