The least understood components of the DNA damage checkpoint are the DNA damage sensors. Genetic studies of Schizosaccharomyces pombe identified six yeast genes, Rad3, Rad17, Rad9, Rad1, Hus1, and Rad26, which encode proteins thought to sense DNA damage and activate the checkpoint-signaling cascade. It has been suggested that Rad9, Rad1 and Hus1 make a heterotrimeric complex forming a PCNA-like structure. In order to carry out structural and biophysical studies of the complex and its associated proteins, the cDNAs encoding full length human Rad9, Rad1 and Hus1 were cloned together into the pET28a vector using a one-step ligation procedure. Here we report successful tri-cistronic cloning, overexpression and purification of this three-protein complex using a single hexa-histidine tag. The trimeric protein complex of Rad9, Rad1 and Hus1 was purified to near homogeneity, yielding approximately 10mg of protein from one liter of Escherichia coli culture.
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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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