Essential role of the iron-sulfur cluster binding domain of the primase regulatory subunit Pri2 in DNA replication initiation
Received date: 09 Dec 2014
Accepted date: 04 Jan 2015
Published date: 01 Apr 2015
Copyright
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.
Key words: primase; Pri2; iron-sulfur cluster; replication initiation
Lili Liu , Mingxia Huang . Essential role of the iron-sulfur cluster binding domain of the primase regulatory subunit Pri2 in DNA replication initiation[J]. Protein & Cell, 2015 , 6(3) : 194 -210 . DOI: 10.1007/s13238-015-0134-8
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