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...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 Pril and a regulatory subunit Pri2. Pri2 is responsible for facilitating optimal RNA primer synthesis by Pril and mediating interaction between Pril and DNA polymerase o 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 sodlA 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.展开更多
文摘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 Pril and a regulatory subunit Pri2. Pri2 is responsible for facilitating optimal RNA primer synthesis by Pril and mediating interaction between Pril and DNA polymerase o 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 sodlA 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.
