DNA primase catalyzes de novo synthesis of a short RNA primer that is further extended by replicative DNA polymerases during initiation of DNA replication. The eukaryotic primase is a heterodimeric enzyme comprising a catalytic subunit Pri1 and a regulatory subunit Pri2. Pri2 is responsible for facilitating optimal RNA primer synthesis by Pri1 and mediating interaction between Pri1 and DNA polymerase α for transition from RNA synthesis to DNA elongation. All eukaryotic Pri2 proteins contain a conserved C-terminal iron-sulfur (Fe-S) cluster-binding domain that is critical for primase catalytic activity in vitro. Here we show that mutations at conserved cysteine ligands for the Pri2 Fe-S cluster markedly decrease the protein stability, thereby causing S phase arrest at the restrictive temperature. Furthermore, Pri2 cysteine mutants are defective in loading of the entire DNA pol α-primase complex onto early replication origins resulting in defective initiation. Importantly, assembly of the Fe-S cluster in Pri2 is impaired not only by mutations at the conserved cysteine ligands but also by increased oxidative stress in the sod1Δ mutant lacking the Cu/Zn superoxide dismutase. Together these findings highlight the critical role of Pri2's Fe-S cluster domain in replication initiation in vivo and suggest a molecular basis for how DNA replication can be influenced by changes in cellular redox state.
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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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Evidence ID | Analyze ID | File | Description |
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