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

Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins

PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication

Investigating the redox properties of human DNA primase

Human DNA primase is a heterodimeric, DNA- dependent RNA polymerase that initiates replication on single- stranded DNA. Primase has been shown previously to contain a [4Fe4S] cluster cofactor in the C terminal domain of its large subunit (p58C) . Here we measure the redox activity of the [4Fe4S] domain of primase (p58C) using electrochem. on a DNA- modified gold electrode. P58C is found to be electrochem. active on duplex DNA substrates with a single- stranded DNA overhang. Binding of p58C to the DNA substrate, moreover, is found to be dependent both on oxidn. state of the [4Fe4S] cluster and on the presence of nucleotide triphosphates (NTPs) . Through these studies, we are exploring how DNA charge transport may be exploited by DNA primase in order to execute the multistep, coordinated process of replication initiation

Optimizing adherence to preexposure and postexposure prophylaxis: the need for an integrated biobehavioral approach

Preexposure prophylaxis (PrEP) and postexposure prophylaxis (PEP) has been shown to be effective in preventing transmission of human immunodeficiency virus (HIV). A dose-response relationship between adherence and HIV transmission is illustrated in the current PrEP literature, and adherence interventions for PrEP may be useful, although currently few effective programs have been developed and tested. There is a paucity of randomized controlled trials testing PEP adherence interventions, and further research is needed. We conclude by proposing the importance of tailoring adherence counseling to address psychosocial factors and mental health stressors that may negatively affect adherence

The Mechanisms for counting and handoff by human DNA primase: a role for the 4Fe-4S cluster?

[No abstract

The Mechanisms for counting and handoff by human DNA primase: a role for the 4Fe-4S cluster?

[No abstract