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.

Charge Transfer between [4Fe4S] Proteins and DNA Is Unidirectional: Implications for Biomolecular Signaling

“…In conclusion, we demonstrate that protein-mediated hole transfer or excess electron transfer between an iron-sulfur cluster and a nucleic acid duplex is always possible in one direction, on a microsecond timescale, irrespective of the specific charge-hopping pathway used to accomplish the CT. This finding solves a main point of debate in the recent literature, 4,[13][14][15] showing that the redox ''communication'' between the [4Fe4S] cluster and nucleic acid is, indeed, possible 15 irrespective of the specific protein pathway involved.…”

“…These timescales (and especially the one for charge transport through M307 only) theoretically support the general validity of the experimental finding that the [4Fe4S] cluster can serve as an effective redox switch to modulate p58c-duplex interactions. 4,15 In fact, our result was obtained for a protein structure different from the mutated one used in O'Brien et al 4 (therefore, a different specific CT pathway in the protein is at play) and is expected to be valid for any iron-sulfur protein with sufficient content of Tyr, Trp, and Met residues. Therefore, hole transfer from an oxidized RNA/DNA duplex to the [4Fe4S] 2+ cluster bound to p58c can occur on biologically relevant timescales, producing the [4Fe4S] 3+ cluster and strengthening the protein-duplex binding (thus preparing the initial state in signaling mechanism 1 of Figure 2A); and hole transfer can also occur from the duplex to p180c (allowed CT process in mechanism 1, which is denoted as step 3 h in Figure 1B).…”

“…The above analysis does not rule out that, in a mutated protein, changes in p58 local folding 4,15 can significantly alter the Tyr positions and therefore the specific route that enables the fastest electron transfer from the iron-sulfur cluster to the nucleic acid.…”

“…3,11 The redox signaling mechanism proposed by O'Brien et al 4 is the subject of debate. 4,[13][14][15] A major criticism of this mechanism arises from the fact that O'Brien et al 4 used a partially misfolded p58c in their study. 16 Use of wild-type p58c should exclude Y345 and Y347 from participating in strong CT pathways between the [4Fe4S] cluster and the duplex 12,17 and would suggest a different explanation for the findings of O'Brien et al 13,14 Furthermore, a different mechanism was proposed for the primer synthesis, in which p58c motion, rather than a charge-transfer mechanism, is responsible for both the termination of the primer synthesis and its loading to Pola.…”

Mutation effects on charge transport through the p58c iron–sulfur protein

“…The charge transport mechanism is the subject of ongoing debate, 9,10,13,14 with questions surrounding the amino acid residues involved in the charge transfer (CT) routes through the p58c protein. 9,[13][14][15] In particular, Y345 has emerged as one of the p58c residues receiving attention in the debate. The functional relevance of Y345 is certainly indicated by the fact that the Y345C mutation is linked to gastric tumors, 16 and ref.…”