Physiological Tolerance to ssDNA Enables Strand Uncoupling during DNA Replication

Ercilla, Amaia; Benada, Jan; Amitash, Sampath; Zonderland, Gijs; Baldi, Giorgio; Somyajit, Kumar; Ochs, Fena; Costanzo, Vincenzo; Lukáš, Jiří; Toledo, Luis I.

doi:10.1016/j.celrep.2020.01.067

Cited by 52 publications

(54 citation statements)

References 53 publications

(60 reference statements)

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“…Helicase-polymerase uncoupling has been described in various checkpoint mutants when exposed to DNA damage or hydroxyurea, the effector kinase of the intra-S-phase checkpoint, Rad53, has been proposed as a key regulator of CMG progression in the absence of replication (Gan et al 2017;Devbhandari and Remus 2020). In our experiments, Rad53 is phosphorylated in the absence of Pol α function in cdc17-1,2-FRB cells, which confirms studies in other eukaryotes showing that the intra-S-phase checkpoint is activated in the presence of aphidicolin or polymerase α inhibition (Byun et al 2005;Ercilla et al 2020). We found that the intra-S-phase checkpoint does not ultimately regulate movement of the CMG complex when polymerase α function is perturbed.…”

Section: Discussionsupporting

confidence: 89%

“…Our results suggest that in addition to impacting replisome progression, the mechanisms that regulate helicase-polymerase uncoupling contribute to replication initiation. Failure to regulate helicase uncoupling at the origin via a "dead man's switch" may trigger replication catastrophe resulting from the generation of excess ssDNA and the sequestration of RPA (Toledo et al 2013;Ercilla et al 2020), thus mechanisms regulating helicase uncoupling from the replisome are likely conserved across eukaryotes and critical for preserving genome stability.…”

Section: Discussionmentioning

confidence: 99%

“…In eukaryotes, helicase-polymerase uncoupling was initially observed in Xenopus extracts when aphidicolin treatment to inhibit polymerases α and δ caused extensive negative supercoiling, indicating that DNA could be unwound by the helicase even in the absence of replication (Walter and Newport 2000;Pacek and Walter 2004). Subsequently, uncoupling of the helicase and at least leading strand polymerization have been observed in multiple contexts that disrupt replication, including polymerase inhibition, DNA damage, and dNTP depletion (Ercilla et al 2020;Nedelcheva et al 2005;Sogo et al 2002;Gan et al 2017;Katou et al 2003;Byun et al 2005). Helicase-polymerase uncoupling typically results in the activation of the intra-S-phase checkpoint (Byun et al 2005).…”

Section: Introductionmentioning

confidence: 99%

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Disruption of origin chromatin structure by helicase activation in the absence of DNA replication

Hoffman

MacAlpine

2021

Preprint

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Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S-phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin which extends up to one kilobase from early, efficient replication origins. The CMG holo-helicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex, but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Disruption of origin chromatin structure by helicase activation in the absence of DNA replication

Hoffman

MacAlpine

2021

Preprint

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“…5A&B). Similarly, a recent report in human cells detected higher levels of RPA-bound ssDNA upon Pol knockdown (47). While Pol hypomorphs in humans show evidence of underreplicated DNA and DSBs, it is unknown if repair of damage due to hypomorphic Pol is repaired by HR or another mechanism in humans (21).…”

Section: Discussionmentioning

confidence: 94%

Limiting DNA polymerase delta alters replication dynamics and leads to a dependence on checkpoint activation and recombination-mediated DNA repair

Koussa

Smith

2019

Preprint

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DNA polymerase delta (Pol ∂) plays several essential roles in eukaryotic DNA replication and repair.At the replication fork, Pol ∂ is responsible for the synthesis and processing of the lagging strand; this role requires Pol ∂ to extend Okazaki fragment primers synthesized by Pol ⍺-primase, and to carry out strand-displacement synthesis coupled to nuclease cleavage during Okazaki fragment termination.Destabilizing mutations in human Pol ∂ subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol ∂ in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze the how the depletion of Pol ∂ impacts replication initiation and elongation in vivo in S. cerevisiae. We determine that Pol ∂ depletion leads to a dependence on checkpoint signaling and recombination-mediated repair for cellular viability. By analyzing nascent lagging-strand products, we observe both a genome-wide change in the establishment and progression of replication forks and a global defect in Pol ∂-mediated Okazaki fragment processing. Additionally, we detect significant lagging-strand synthesis by the leading-strand polymerase (Pol ɛ) in late-replicating regions of the genome when Pol ∂ is depleted. Together, our results are consistent with the presence of least two Pol ∂ complexes in the eukaryotic replisome under normal conditions. We propose that replisomes with sub-stoichiometric Pol ∂ are defective due to competition between nascent lagging-strand primers and terminating Okazaki fragments, and that this competition underlies both the replication and genome stability defects observed when cellular levels of Pol ∂ fall below a critical threshold. Supplementary figure 1 (associated with figure 1). LEGENDS TO SUPPLEMENTARY FIGURES Further characterization of Pol3 depletion kinetics and the effect of depletion on growth rate.A. Western blot against Pol3-9Myc or TIR1-9Myc in asynchronous cultures of the POL3-AID strain following 30 minutes of treatment with the indicated concentration of IAA B. Growth rates of POL3-AID cells in liquid culture. Data were calculated from three replicates.C. DNA content measured by flow cytometry for logarithmically growing POL3-AID cells treated with the indicated concentration of IAA for 2hSupplementary figure 2 (associated with figure 3). Effects of Pol3 depletion on replication origin firing in individual replicates.A-F. Analysis of replication origin firing efficiency as shown for pooled replicates in Fig. 3B,D&E, separated by individual biological replicate strain as indicated. **** denotes p < 0.0001 by Kolmogorov-Smirnov test (A,D) or Wilcoxon signed rank test (B-C, E-F) for a change in the distribution or mean of the data, respectively. Supplementary figure 3 (associated with figure 4) Samples used for analysis of replication-fork speed in figure 4.DNA content measured by flow cytometry for the samples shown in Fig. 4A. Red timepoints were sequenced for both 0 and 1 mM IAA, and blue timepoints for 1 mM only. figure 4 (associated with figure 5) Addi...

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“…Although TLS-induced mutations and lesion bypass cannot be overlooked as providing a selective advantage for tumorigenesis and/or chemoresistance, TLS-induced RGS should also be considered. First, RGS mitigates replication gaps that cause replication catastrophe, a situation in which ssDNA becomes exposed to nucleases when ssDNA exceeds protection by the ssDNA binding protein, RPA [101]. Conceivably, oncogene-induced senescence is also linked to replication catastrophe so that oncogenic transformation requires either RGS by TLS or higher RPA levels.…”

Section: Tls-induced Mutagenesis Vs Rgs In Cancer Developmentmentioning

confidence: 99%

Targeting translesion synthesis (TLS) to expose replication gaps, a unique cancer vulnerability

Nayak

Calvo

Cantor

2021

Expert Opinion on Therapeutic Targets

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Physiological Tolerance to ssDNA Enables Strand Uncoupling during DNA Replication

Cited by 52 publications

References 53 publications

Disruption of origin chromatin structure by helicase activation in the absence of DNA replication

Disruption of origin chromatin structure by helicase activation in the absence of DNA replication

Limiting DNA polymerase delta alters replication dynamics and leads to a dependence on checkpoint activation and recombination-mediated DNA repair

Targeting translesion synthesis (TLS) to expose replication gaps, a unique cancer vulnerability

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