Reference: Salay LE, et al. (2022) Modification of the 4Fe-4S Cluster Charge Transport Pathway Alters RNA Synthesis by Yeast DNA Primase. Biochemistry 61(11)

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Abstract


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

Reference Type
Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.
Authors
Salay LE, Blee AM, Raza MK, Gallagher KS, Chen H, Dorfeuille AJ, Barton JK, Chazin WJ
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