Yeast require redox switching in DNA primase

O’Brien, Elizabeth; Salay, Lauren E.; Friedman, Katherine L.; Chazin, Walter J.; Barton, Jacqueline K.

doi:10.1073/pnas.1810715115

Cited by 16 publications

(36 citation statements)

References 46 publications

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“…20 (Figure 3) In our HEPES electrochemistry buffer (20 mM HEPES, pH 7.2, 150 mM NaCl, 5% glycerol), the signal is centered near 160 mV vs NHE. This signal is within the range expected for DNA-processing [4Fe4S] enzymes cycling between the [4Fe4S] 2+ and [4Fe4S] 3+ states, 19,20,24–29 and is similar to the reported values for human and yeast p58C 20,27 in the presence of DNA and NTPs.…”

Section: Resultssupporting

confidence: 86%

“…The midpoint potential of primase in the presence of DNA and NTPs (160 ± 4 mV vs NHE) is slightly higher than the midpoint potentials observed for human and yeast p58C in the presence of DNA and NTPs, which is near 150 mV vs NHE. 20,27 This shift may be due to an increased amount of insulating protein matrix surrounding full-length primase as compared to p58C, which promotes a higher reduction potential. 32 Binding of the DNA polyanion and negatively charged NTPs, importantly, still shifts the cluster potential of full-length primase into the physiological range for signaling activity.…”

Section: Resultsmentioning

confidence: 99%

“…The p58C domain of human and yeast DNA primase has been demonstrated electrochemically to undergo DNA-mediated redox switching between an oxidized, [4Fe4S] 3+ state and a resting, [4Fe4S] 2+ redox state. , The oxidized [4Fe4S] 3+ p58C is tightly bound to DNA, whereas the reduced [4Fe4S] 2+ p58C is only loosely bound to DNA. A similar switch is evident in the base excision repair protein, Endonuclease III, which contains a [4Fe4S] cluster; here binding to the DNA polyanion shifts the [4Fe4S] 3+/2+ redox potential so that the oxidized [4Fe4S] 3+ form binds DNA 500-fold more tightly than the [4Fe4S] 2+ form. , This redox switch in p58C appears to regulate primase activity and primer product distribution, yet the electrochemical behavior of the full primase enzyme, relative to that of p58C, has yet to be reported.…”

Section: Introductionmentioning

confidence: 99%

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Substrate Binding Regulates Redox Signaling in Human DNA Primase

et al. 2018

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Generation of daughter strands during DNA replication requires the action of DNA primase to synthesize an initial short RNA primer on the single-stranded DNA template. Primase is a heterodimeric enzyme containing two domains whose activity must be coordinated during primer synthesis: an RNA polymerase domain in the small subunit (p48) and a [4Fe4S] cluster-containing C-terminal domain of the large subunit (p58C). Here we examine the redox switching properties of the [4Fe4S] cluster in the full p48/p58 heterodimer using DNA electrochemistry. Unlike with isolated p58C, robust redox signaling in the primase heterodimer requires binding of both DNA and NTPs; NTP binding shifts the p48/p58 cluster redox potential into the physiological range, generating a signal near 160 mV vs NHE. Preloading of primase with NTPs enhances catalytic activity on primed DNA, suggesting that primase configurations promoting activity are more highly populated in the NTP-bound protein. We propose that p48/p58 binding of anionic DNA and NTPs affects the redox properties of the [4Fe4S] cluster; this electrostatic change is likely influenced by the alignment of primase subunits during activity because the configuration affects the [4Fe4S] cluster environment and coupling to DNA bases for redox signaling. Thus, both binding of polyanionic substrates and configurational dynamics appear to influence [4Fe4S] redox signaling properties. These results suggest that these factors should be considered generally in characterizing signaling networks of large, multisubunit DNA-processing [4Fe4S] enzymes.

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Substrate Binding Regulates Redox Signaling in Human DNA Primase

et al. 2018

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“…We recently observed that the redox-driven DNA binding switch is conserved in yeast as well as human primase (128). On DNA electrodes, oxidized [4Fe4S] 3+ p58C is tightly bound and redox-active, whereas reduced [4Fe4S] 2+ p58C is loosely associated with DNA and redox-inert in yeast and human systems.…”

Section: [4fe4s] Enzymes In Eukaryotic Dna Replicationmentioning

confidence: 99%

Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication

Barton

Silva

O’Brien

2019

Annu. Rev. Biochem.

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Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox cofactor in biology. We find using DNA electrochemistry that binding of the DNA polyanion promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to ~ 500-fold increase in DNA binding affinity for the oxidized [4Fe4S]3+ cluster versus the reduced [4Fe4S]2+ cluster. This redox switch can be activated from a distance using DNA charge transport chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated with possible roles for the redox switch, highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. Thus we describe redox signaling interactions of DNA-processing [4Fe4S] enzymes as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.

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“…The cluster is essential for initiating primer synthesis in vitro ( Klinge et al 2007 ; Weiner et al 2007 ) and in vivo ( Liu and Huang 2015 ). Actually, the redox state of the [4Fe-4S] cluster functions as a reversible switch for DNA binding (“redox switching”) as shown in vitro ( Liu and Huang 2015 ); this allows primase function by fine-tuning protein binding to DNA in yeast and human cells despite pretty strong structural dissimilarities between the two proteins ( O'Brien et al 2018 ). More broadly, a cluster-dependent redox regulation of B-type DNA polymerases has been extensively explored by the Barton’s laboratory [23–26], revealing the major role of redox regulation of DNA polymerases activities in vivo , through oxidation/reduction of the cluster.…”

Section: Introductionmentioning

confidence: 99%

Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae

Chanet

Baïlle

Golinelli-Cohen

et al. 2021

G3 Genes|Genomes|Genetics

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B-type eukaryotic polymerases contain a [4Fe-4S] cluster in their C-terminus domain, whose role is not fully understood yet. Among them, DNA polymerase delta (Polδ) plays an essential role in chromosomal DNA replication, mostly during lagging strand synthesis. Previous in vitro work suggested that the Fe-S cluster in Polδ is required for efficient binding of the Pol31 subunit, ensuring stability of the Polδ complex. Here, we analyzed the in vivo consequences resulting from an impaired coordination of the Fe-S cluster in Polδ. We show that a single substitution of the very last cysteine coordinating the cluster by a serine is responsible for the generation of massive DNA damage during S phase, leading to checkpoint activation, requirement of homologous recombination for repair, and ultimately to cell death when the repair capacities of the cells are overwhelmed. These data indicate that impaired Fe-S cluster coordination in Polδ is responsible for aberrant replication. More generally, Fe-S in Polδ may be compromised by various stress including anti-cancer drugs. Possible in vivo Polδ Fe-S cluster oxidation and collapse may thus occur, and we speculate this could contribute to induced genomic instability and cell death, comparable to that observed in pol3-13 cells.

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Yeast require redox switching in DNA primase

Cited by 16 publications

References 46 publications

Substrate Binding Regulates Redox Signaling in Human DNA Primase

Substrate Binding Regulates Redox Signaling in Human DNA Primase

Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication

Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae

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