Molecular basis for PrimPol recruitment to replication forks by RPA

DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multi-functional replicative enzyme called Primase-Polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPolʼs recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPolʼs mode of recruitment to stalled forks to facilitate repriming and restart

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

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

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

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

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

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

Functional role for the [4Fe4S] cluster in human DNA primase as a redox switch using DNA charge transport

DNA-mediated charge transport (DNA CT) provides an avenue for long-range, rapid signaling between redox-active moieties coupled into duplex DNA. Several DNA-processing enzymes have moreover been shown to contain [4Fe4S] clusters, common redox cofactors. Eukaryotic DNA primase, the heterodimeric enzyme responsible for initiating DNA replication, contains a [4Fe4S] cluster in the C-terminal domain of the large subunit (p58C). Primase synthesizes a short RNA primer on a single-stranded DNA template and subsequently hands this template off to DNA polymerase α, another [4Fe4S] protein, through a mechanism which is unclear. Here we show electrochem. evidence that the [4Fe4S] cluster in the p58C domain of human DNA primase performs redox chem. on DNA, cycling reversibly between a tightly DNA-bound, oxidized [4Fe4S]^(3+) state, and a loosely assocd., reduced [4Fe4S]^(2+) state. We demonstrate through structural, biochem., and electrochem. comparisons of wild type and mutant p58C that the redox switch is mediated by a pathway of tyrosine residues between the cluster and bound DNA. Charge transfer pathway mutations in full-length primase, addnl., abrogate initiation of primer synthesis on single-stranded DNA but do not affect nucleotide polymn. We further compare primer elongation on a well-matched and mismatched DNA template, showing that a single-base mismatch in the nascent primer inhibits primase termination. Thus primer termination appears to be gated by mismatch-sensitive DNA charge transport. Based on our exptl. evidence, we propose a model in which electron transfer between [4Fe4S] clusters, gated by DNA-mediated charge transport, regulates DNA binding and substrate handoff between primase and polymerase α to begin replication

Functional role for the [4Fe4S] cluster in human DNA primase as a redox switch using DNA charge transport

DNA-mediated charge transport (DNA CT) provides an avenue for long-range, rapid signaling between redox-active moieties coupled into duplex DNA. Several DNA-processing enzymes have moreover been shown to contain [4Fe4S] clusters, common redox cofactors. Eukaryotic DNA primase, the heterodimeric enzyme responsible for initiating DNA replication, contains a [4Fe4S] cluster in the C-terminal domain of the large subunit (p58C). Primase synthesizes a short RNA primer on a single-stranded DNA template and subsequently hands this template off to DNA polymerase α, another [4Fe4S] protein, through a mechanism which is unclear. Here we show electrochem. evidence that the [4Fe4S] cluster in the p58C domain of human DNA primase performs redox chem. on DNA, cycling reversibly between a tightly DNA-bound, oxidized [4Fe4S]^(3+) state, and a loosely assocd., reduced [4Fe4S]^(2+) state. We demonstrate through structural, biochem., and electrochem. comparisons of wild type and mutant p58C that the redox switch is mediated by a pathway of tyrosine residues between the cluster and bound DNA. Charge transfer pathway mutations in full-length primase, addnl., abrogate initiation of primer synthesis on single-stranded DNA but do not affect nucleotide polymn. We further compare primer elongation on a well-matched and mismatched DNA template, showing that a single-base mismatch in the nascent primer inhibits primase termination. Thus primer termination appears to be gated by mismatch-sensitive DNA charge transport. Based on our exptl. evidence, we propose a model in which electron transfer between [4Fe4S] clusters, gated by DNA-mediated charge transport, regulates DNA binding and substrate handoff between primase and polymerase α to begin replication