Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity

Syed, Aleem; Filandr, Frantisek; Patterson-Fortin, Jeffrey; Bacolla, Albino; Ravindranathan, Ramya; Zhou, Jia; McDonald, Drew T; Albuhluli, Mohammed E; Verway-Cohen, Amy; Newman, Joseph A; Tsai, Miaw-Sheue; Jones, Darin E; Schriemer, David C; D’Andrea, Alan D; Tainer, John A

doi:10.1093/nar/gkad727

Cited by 7 publications

(7 citation statements)

References 86 publications

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“…To test this automation approach, we applied it to a manually curated dataset collected in DIA mode but analyzed in the conventional MS 1 -only fashion 39 . Pol ϴ, a DNA polymerase, is a cancer drug target.…”

Section: Resultsmentioning

confidence: 99%

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology

Filandr,

Sarpe,

Raval

et al. 2024

Nat Commun

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We present a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-curation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-curation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.

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Section: Resultsmentioning

confidence: 99%

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology

Filandr,

Sarpe,

Raval

et al. 2024

Nat Commun

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“…However, as the compound equally affects the control Firefly luciferase signal, we cannot conclude that the effect is specific and possibly highlights its potentially promiscuous pharmacology ( 46 ). Modulation of Polθ-mediated MMEJ by novobiocin may however reflect a more complicated mechanism of action not captured in the extrachromosomal assay format ( 48 ) and/or one that is revealed in a different context such as a chromosomally integrated MMEJ reporter system ( 45 ).…”

Section: Discussionmentioning

confidence: 99%

Quantitative, titratable and high-throughput reporter assays to measure DNA double strand break repair activity in cells

Rajendra,

Grande,

Mason

et al. 2023

Nucleic Acids Research

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Repair of DNA damage is essential for the maintenance of genome stability and cell viability. DNA double strand breaks (DSBs) constitute a toxic class of DNA lesion and multiple cellular pathways exist to mediate their repair. Robust and titratable assays of cellular DSB repair (DSBR) are important to functionally interrogate the integrity and efficiency of these mechanisms in disease models as well as in response to genetic or pharmacological perturbations. Several variants of DSBR reporters are available, however these are often limited by throughput or restricted to specific cellular models. Here, we describe the generation and validation of a suite of extrachromosomal reporter assays that can efficiently measure the major DSBR pathways of homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single strand annealing (SSA). We demonstrate that these assays can be adapted to a high-throughput screening format and that they are sensitive to pharmacological modulation, thus providing mechanistic and quantitative insights into compound potency, selectivity, and on-target specificity. We propose that these reporter assays can serve as tools to dissect the interplay of DSBR pathway networks in cells and will have broad implications for studies of DSBR mechanisms in basic research and drug discovery.

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“…Asynchronous POLQ -/-HF1 and HCT116 cells each showed a reproducible, significant, but partial loss of deletions as well as other types of SVs relative to wild-type (Figures 5D, 5E, S9A and S9B). Deletion SV reduction was again partial in HCT116 asynchronous or M-phase cells treated with the POLQ inhibitors ART558 48 or novobiocin (NVB) 49,50 (Figures 5E and 5F). In contrast, APH-treated POLQ -/-HCT116 Mphase cells showed baseline levels of SV formation with no apparent induction by APH (Figures 5F and S9C).…”

Section: Chemical Polq Inhibition and Polq Knockout Differentially Im...mentioning

confidence: 99%

POLQ mediates replication-stress induced structural variant formation throughout common fragile sites during mitosis

Wilson,

Ahmed,

Winningham

et al. 2024

Preprint

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Genomic structural variants (SVs) greatly impact human health and disease, but much is unknown about their generative mechanisms, especially for the large class of nonrecurrent alterations. Common fragile sites (CFSs) are unstable loci that provide a model for SV formation, especially large deletions, under replication stress. We studied SV junction formation as it occurred in cells by applying error-minimized capture sequencing to CFS DNA harvested during replication stress. SV junctions formed throughout CFS genes at a 5-fold higher rate after cells passed from G2 into M-phase. Neither SV formation nor CFS expression depended on mitotic DNA synthesis (MiDAS), an error-prone form of conservative replication active at CFSs. Instead, analysis of tens of thousands ofde novoSV junctions combined with DNA repair pathway inhibition revealed a primary role for DNA polymerase theta (POLQ)-mediated end-joining (TMEJ) in M-phase SV formation. We propose an important role for TMEJ in nonrecurrent SV formation genome wide.

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Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity

Cited by 7 publications

References 86 publications

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology

Quantitative, titratable and high-throughput reporter assays to measure DNA double strand break repair activity in cells

POLQ mediates replication-stress induced structural variant formation throughout common fragile sites during mitosis

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