Modification of the 4Fe-4S Cluster Charge Transport Pathway Alters RNA Synthesis by Yeast DNA Primase

• DOI: 10.1021/acs.biochem.2c00100
• Publication Date: 2022-05-26T21:19:46Z
• Authors: Lauren E. Salay; Alexandra M. Blee; Md Kausar Raza; Kaitlyn S. Gallagher; Huiqing Chen; Andrew J. Dorfeuille; Jacqueline K. Barton; Walter J. Chazin
• Keywords: Primase; Primer (cosmetics)

DNA synthesis during replication begins with the generation of an ∼10-nucleotide primer by primase. Primase contains a redox-active 4Fe-4S cluster in C-terminal domain p58 subunit (p58C). The redox state this can be modulated via transport charge through protein and substrate (redox switching); changes alter ability p58C to associate its substrate. efficiency switching altered mutating tyrosine residues that bridge nucleic acid binding site. Here, we report effects bridging tyrosines phenylalanines yeast p58C. High-resolution crystal structures show these mutations, even six simultaneously mutated, do not perturb three-dimensional structure protein. In contrast, measurements electrochemical properties on DNA-modified electrodes containing multiple phenylalanine mutations reveal deficiencies their engage transport. Significantly, loss activity correlates decreased primase activity. While single-site mutants showed modest decreases compared wild-type primase, exhibited 10-fold or greater decrease. Thus, many possible tyrosine-mediated pathways for exist, but inhibiting together diminishes generate primers. These results support model which is essential