title: Essential role of the iron-sulfur cluster binding domain of the primase regulatory subunit Pri2 in DNA replication initiation.
abstract: 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.
authors: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA.
authors: Lili
authors: L
authors: Liu
authors: Mingxia
authors: M
authors: Huang
mesh_terms: D000595:Amino Acid Sequence; D002453:Cell Cycle; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D007501:Iron / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D018384:Oxidative Stress; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013455:Sulfur / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; D007506:Iron-Sulfur Proteins; D013455:Sulfur; D007501:Iron; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase; D003545:Cysteine
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