Response to Comments on "The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport"

Abstract

Baranovskiy et al and Pellegrini argue that, based on structural data, the path for charge transfer through the [4Fe4S] domain of primase is not feasible. Our manuscript presents electrochemical data directly showing charge transport through DNA to the [4Fe4S] cluster of a primase p58C construct and a reversible switch in the DNA-bound signal with oxidation/reduction, which is inhibited by mutation of three tyrosine residues. Although the dispositions of tyrosines differ in different constructs, all are within range for microsecond electron transfer.

Fig. 1. Wild-type (WT) p48/p58 versus p48/p58Y309F activity on ssDNA

(A) Gel separation of products for primase initiation assay comparing WT and charge transfer–deficient primase. (B) Product quantifications for p48/p58Y309F, with WT primase (p48/p58) products normalized to one. Values shown are the mean of n = 3 trials; error bars represent standard deviation. *, 0.001 ≤ P < 0.005; **, P < 0.001; Student’s t test. All activity assays were performed under anaerobic conditions. Reactions contained 400 nM primase variant, 250 nM ssDNA, 188 μM uridine triphosphate (UTP), 112 μM cytidine triphosphate (CTP), 1 μM α⁻³²P adenosine triphosphate (ATP) in 50 mM Tris, pH 8.0, 3 mM MgCl₂.