Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency

Cong, Ke; Peng, Min; Kousholt, Arne Nedergaard; Lee, Wei Ting C.; Lee, Silviana; Nayak, Sumeet; Krais, John J.; VanderVere-Carozza, Pamela S.; Pawelczak, Katherine S.; Calvo, Jennifer A.; Panzarino, Nicholas J.; Turchi, John J.; Johnson, Neil; Jonkers, Jos; Rothenberg, Eli; Cantor, Sharon B.

doi:10.1016/j.molcel.2021.06.011

Cited by 178 publications

(175 citation statements)

References 135 publications

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“…Interestingly, recently published data indicate that defective TLS enhances the formation of post-replicative ssDNA gaps by favoring PRIMPOL-dependent repriming beyond damaged bases (Nayak et al, 2020; Quinet et al, 2021). Moreover, formation of such ssDNA gaps have been shown to cause strong sensitivity to replicative stress (Cong et al, 2021; Panzarino et al, 2021). It therefore seems likely that ssDNA gap formation behind RF underlie the strong representation of TLS polymerases, and regulators thereof, in our screen.…”

Section: Discussionmentioning

confidence: 99%

A genome-wide screen identifies SCAI as a modulator of the UV-induced replicative stress response in human cells

Lemay¹,

St-Hilaire²,

Gezzar-Dandashi³

et al. 2022

Preprint

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Helix-destabilizing DNA lesions induced by environmental mutagens such as UV light cause genomic instability by strongly blocking the progression of DNA replication forks (RF). At blocked RF, single-stranded DNA (ssDNA) accumulates and is rapidly bound by Replication Protein A (RPA) complexes. Such stretches of RPA-ssDNA constitute platforms for recruitment/activation of critical factors that promote DNA synthesis restart. However, during periods of severe replicative stress, RPA availability may become limiting due to inordinate sequestration of this multifunctional complex on ssDNA, thereby negatively impacting multiple vital RPA-dependent processes. Here, we performed a genome-wide screen to identify factors which restrict the accumulation of RPA-ssDNA during UV-induced replicative stress. While this approach revealed some expected “hits” acting in pathways such as nucleotide excision repair, translesion DNA synthesis, and the intra-S phase checkpoint, it also identifed SCAI, whose role in the replicative stress response was previously unappreciated. Upon UV exposure, SCAI knock-down caused elevated accumulation of RPA-ssDNA during S phase, accompanied by reduced cell survival and compromised RF progression. These effects were independent of the previously reported role of SCAI in 53BP1-dependent DNA double-strand break repair. We also found that SCAI colocalized with stalled RF, and that its depletion promoted nascent DNA degradation. Finally, we (i) provide evidence that EXO1 is the major nuclease underlying ssDNA formation and consequent DNA replication defects in SCAI knockout cells and, consistent with this, (ii) demonstrate that SCAI inhibits EXO1 activity on a ssDNA gap in vitro . Taken together, our data establish SCAI as a novel regulator of the replicative stress response in human cells.

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Section: Discussionmentioning

confidence: 99%

A genome-wide screen identifies SCAI as a modulator of the UV-induced replicative stress response in human cells

Lemay¹,

St-Hilaire²,

Gezzar-Dandashi³

et al. 2022

Preprint

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“…Data from recent studies indicate that LIG3 is present at replication forks (Arakawa and Iliakis, 2015;Hanzlikova et al, 2018;Sriramachandran et al, 2020;Cong et al, 2021). Therefore, we next investigated whether LIG3 localizes to sites of DNA replication marked by 5-ethynyl-2'deoxyuridine (EdU) incorporation, in the absence of DNA damage induction.…”

Section: Lig3 Is Required At Replication Forks In Brca1-deficient Cells Treated With Parpimentioning

confidence: 99%

Loss of Nuclear DNA Ligase III Reverts PARP Inhibitor Resistance in BRCA1/53BP1 Double-deficient Cells by Exposing ssDNA Gaps

Dias

Tripathi

Heijden

et al. 2021

Preprint

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Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1, identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 is required for PARPi-induced fork acceleration in BRCA1-deficient cells and that LIG3 loss increases fork asymmetry. Furthermore, LIG3 depletion in BRCA1-deficient cells results in an increase in ssDNA gaps behind the replication forks, resulting in accumulation of chromosomal abnormalities. We also report that high expression of LIG3 in patients with invasive breast cancer correlates in with poorer overall survival, rendering LIG3 as a potential therapeutic target for enhancing PARPi sensitivity.

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“…More recently, a flurry of studies in human cells demonstrated the existence of DSGs in newly synthesized DNA by employing EdU pulse-labelling experiments coupled to super-resolution microscopy [ 69 ] or by employing a modified DNA fiber assay coupled to S1-nuclease digestion [ 11 , 60 , 69 , 89 , 90 , 91 ]. Single-stranded DSGs within DNA fibers are sensitive to S1-nuclease activity and can thus be indirectly measured by the extent of DNA fragmentation [ 92 ].…”

Section: Hr As An Intrinsic Safeguard Of Dna Replication In Mammalian Somatic Cellsmentioning

confidence: 99%

Homologous Recombination as a Fundamental Genome Surveillance Mechanism during DNA Replication

Spies

Polasek-Sedlackova

Lukáš

et al. 2021

Genes

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Accurate and complete genome replication is a fundamental cellular process for the proper transfer of genetic material to cell progenies, normal cell growth, and genome stability. However, a plethora of extrinsic and intrinsic factors challenge individual DNA replication forks and cause replication stress (RS), a hallmark of cancer. When challenged by RS, cells deploy an extensive range of mechanisms to safeguard replicating genomes and limit the burden of DNA damage. Prominent among those is homologous recombination (HR). Although fundamental to cell division, evidence suggests that cancer cells exploit and manipulate these RS responses to fuel their evolution and gain resistance to therapeutic interventions. In this review, we focused on recent insights into HR-mediated protection of stress-induced DNA replication intermediates, particularly the repair and protection of daughter strand gaps (DSGs) that arise from discontinuous replication across a damaged DNA template. Besides mechanistic underpinnings of this process, which markedly differ depending on the extent and duration of RS, we highlight the pathophysiological scenarios where DSG repair is naturally silenced. Finally, we discuss how such pathophysiological events fuel rampant mutagenesis, promoting cancer evolution, but also manifest in adaptative responses that can be targeted for cancer therapy.

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Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency

Cited by 178 publications

References 135 publications

A genome-wide screen identifies SCAI as a modulator of the UV-induced replicative stress response in human cells

A genome-wide screen identifies SCAI as a modulator of the UV-induced replicative stress response in human cells

Loss of Nuclear DNA Ligase III Reverts PARP Inhibitor Resistance in BRCA1/53BP1 Double-deficient Cells by Exposing ssDNA Gaps

Homologous Recombination as a Fundamental Genome Surveillance Mechanism during DNA Replication

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