Research Article

The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport

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Science  24 Feb 2017:
Vol. 355, Issue 6327, eaag1789
DOI: 10.1126/science.aag1789

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DNA charged with regulating replication

DNA can transport electrical charge over long distances and has the potential to act as a signaling system. The iron-sulfur complex [4Fe4S] found in some proteins is known to be involved in redox reactions. The eukaryotic DNA primase is involved in DNA replication and contains a [4Fe4S] cluster that is required for its RNA primer synthesis activity. O'Brien et al. show that the [4Fe4S] cluster in DNA primase can regulate the protein's DNA binding activity through DNA-mediated charge transfer. This in turn plays a role in primer initiation and length determination.

Science, this issue p. eaag1789

Structured Abstract

INTRODUCTION

DNA charge transport provides an avenue for rapid, long-range signaling between redox-active moieties coupled into the DNA duplex. Several enzymes integral to eukaryotic DNA replication contain [4Fe4S] clusters, common redox cofactors. DNA primase, the enzyme responsible for initiating replication on single-stranded DNA, is a [4Fe4S] protein. Primase synthesizes short RNA primers of a precise length before handing off the primed DNA template to DNA polymerase α, another [4Fe4S] enzyme. The [4Fe4S] cluster in primase is required for primer synthesis, but its underlying chemistry has not been established. Moreover, what orchestrates primer handoff between primase and DNA polymerase α is not well understood.

RATIONALE

DNA-mediated electrochemistry, single-atom mutations, structural characterization, and activity studies together elucidate the functional role of the [4Fe4S] cluster in DNA primase. DNA electrochemistry under anaerobic conditions allows precise control over cluster oxidation state and provides insight into the redox activity of the [4Fe4S] cluster domain, p58C, bound to DNA, in the oxidized [4Fe4S]3+ state as compared to the reduced [4Fe4S]2+ state. Structural and biochemical studies complement the electrochemical assays by using charge transfer–deficient mutants to establish how activity is affected by cluster redox state. Enzymatic studies examine how both the redox state of the [4Fe4S] cluster and DNA charge transport regulate primase activity and primer-template truncation as the first step in primase handoff to polymerase α.

RESULTS

Using DNA-mediated electrochemistry, we compared the DNA-bound redox activity of the DNA primase [4Fe4S] cluster domain p58C in the oxidized [4Fe4S]3+ state and in the reduced [4Fe4S]2+ state. We found that p58C binds DNA tightly and is coupled into the duplex for redox activity in the oxidized state but is more loosely associated with the DNA and redox-inactive in the reduced state. Moreover, the redox signal in cyclic voltammetry scans of the electrochemically oxidized p58C bound to the DNA electrode disappeared upon electrochemical reduction but was regenerated repeatedly with subsequent electrochemical oxidation. Thus, the redox state of the [4Fe4S] cluster in DNA primase functions as a reversible switch for DNA binding.

We investigated a possible charge transfer pathway through the p58C protein matrix, between bound DNA and the [4Fe4S] cluster; such a pathway is necessary to mediate the redox switch reaction. Three conserved tyrosine residues positioned between the [4Fe4S] cluster and the p58C DNA-binding interface were selected as possible components in a charge transfer pathway, as tyrosines are readily ionized and often mediate electron transfer reactions through proteins. To test whether electron tunneling through the p58C matrix affects this switch and is mediated by the conserved tyrosines, we designed and isolated three single-atom, tyrosine-to-phenylalanine p58C mutants. DNA-mediated electrochemistry and biophysical characterization showed that these tyrosine mutations inhibit the redox switch but cause no change in the structure of p58C or in its ability to bind DNA. Tyrosine mutations compromising the charge transfer pathway were then engineered into full-length primase. Anaerobic activity assays showed that primase mutants deficient in the redox switch display impaired initiation activity, but not elongation activity, relative to wild-type protein. The redox switch in primase is thus essential for initiation on single-stranded DNA but not for nucleotide polymerization.

We further found that when a base pair mismatch is introduced into the growing primer, inhibiting DNA charge transport through the nascent duplex, primase loses the ability to truncate products. On the basis of these findings, we propose that the redox switch reaction altering the oxidation state of the [4Fe4S] cluster (i) chemically regulates primase binding to DNA and (ii) facilitates DNA-mediated redox signaling for primer truncation and handoff to polymerase α.

CONCLUSION

We demonstrate that the oxidation state of the [4Fe4S] cluster in DNA primase acts as a reversible on/off switch for DNA binding. Moreover, both the conserved charge transfer pathway through primase and DNA charge transport chemistry play crucial roles in regulating primer synthesis. Our findings provide a chemical basis for understanding the precise regulation of primase activity, using DNA charge transport for redox signaling of [4Fe4S] clusters, and support a fundamentally new redox switch model for substrate handoff. Such redox signaling by [4Fe4S] clusters may play a wider role in polymerase enzymes to coordinate eukaryotic DNA replication.

The redox switch in DNA primase regulates initiation and termination of priming.

(Left) Electrochemically altering the redox state of the [4Fe4S] cluster in the p58C domain of DNA primase on a DNA electrode facilitates reversible switching between loosely associated, redox-inactive p58C and DNA-bound, redox-active p58C. (Right) Model for primase product truncation, where primer-template handoff to the [4Fe4S] signaling partner, polymerase α in vivo, is regulated by DNA charge transport.

Abstract

DNA charge transport chemistry offers a means of long-range, rapid redox signaling. We demonstrate that the [4Fe4S] cluster in human DNA primase can make use of this chemistry to coordinate the first steps of DNA synthesis. Using DNA electrochemistry, we found that a change in oxidation state of the [4Fe4S] cluster acts as a switch for DNA binding. Single-atom mutations that inhibit this charge transfer hinder primase initiation without affecting primase structure or polymerization. Generating a single base mismatch in the growing primer duplex, which attenuates DNA charge transport, inhibits primer truncation. Thus, redox signaling by [4Fe4S] clusters using DNA charge transport regulates primase binding to DNA and illustrates chemistry that may efficiently drive substrate handoff between polymerases during DNA replication.

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