Essential role of the iron-sulfur cluster binding domain of the primase regulatory subunit Pri2 in DNA replication initiation.
10.1007/s13238-015-0134-8
- Author:
Lili LIU
1
;
Mingxia HUANG
Author Information
1. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Cell Cycle;
Cell Proliferation;
Chromatin Immunoprecipitation;
Cysteine;
genetics;
metabolism;
DNA Primase;
genetics;
metabolism;
DNA Replication;
DNA, Fungal;
genetics;
DNA-Directed DNA Polymerase;
metabolism;
Immunoblotting;
Immunoprecipitation;
Iron;
metabolism;
Iron-Sulfur Proteins;
metabolism;
Molecular Sequence Data;
Mutation;
genetics;
Oxidative Stress;
Protein Binding;
Saccharomyces cerevisiae;
genetics;
growth & development;
metabolism;
Sequence Homology, Amino Acid;
Sulfur;
metabolism
- From:
Protein & Cell
2015;6(3):194-210
- CountryChina
- Language:English
-
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.
