Essential role of the iron-sulfur cluster binding domain of the primase regulatory subunit Pri2 in DNA replication initiation
Protein & Cell
;
(12): 194-210, 2015.
Article
in English
| WPRIM
| ID: wpr-757602
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Protein Binding
/
Saccharomyces cerevisiae
/
Sulfur
/
DNA, Fungal
/
Molecular Sequence Data
/
Immunoblotting
/
Cell Cycle
/
Amino Acid Sequence
/
Sequence Homology, Amino Acid
/
Oxidative Stress
Language:
English
Journal:
Protein & Cell
Year:
2015
Type:
Article
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