Topoisomerase Assays

Nitiss, John L.; Kiianitsa, Kostantin; Sun, Yilun; Nitiss, Karin C.; Maizels, Nancy

doi:10.1002/cpz1.250

Cited by 21 publications

(20 citation statements)

References 118 publications

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“…Given the heterogeneity of the non-enzymatic DPCs induced by FA, the repair of those DPCs remains only partially known. Understanding the protein identity of as well as chromosome regions, DNA transactions and DNA structures involved in the DPCs is therefore important to study 34, 35 non-enzymatic DPC repair. A major challenge to profiling non-enzymatic DPCs is their purification.…”

Section: Resultsmentioning

confidence: 99%

Flap endonuclease 1 repairs DNA-protein crosslinks via ADP-ribosylation

Sun,

Jenkins,

El Touny

et al. 2023

Preprint

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DNA-protein crosslinks (DPCs) are among the most ubiquitous and detrimental DNA lesions which arise from exposure to metabolic stresses, drugs, or crosslinking agents such as formaldehyde (FA). FA is a cellular by-product of methanol metabolism, histone demethylation, lipid peroxidation as well as environmental pollutants. Failure to repair FA-induced DPCs blocks nearly all chromatin-based processes including replication and transcription, leading to immunodeficiencies, neurodegeneration, and cancer. Yet, it remains largely unknown how the cell repairs DPCs. The study of DPC repair is impeded by our incomprehension of the types of proteins crosslinked by FA due to the lack of techniques to identify the DPCs. Here, we designed a novel bioassay to profile FA-induced DPCs by coupling cesium chloride differential ultracentrifugation with HPLC-mass spectrometry (MS). Using the method, we revealed the proteome of FA-induced DPCs in human cells and found that the most abundant proteins that form DPCs are PARP1, topoisomerases I and II, methyltransferases, DNA and RNA polymerases, histones, as well as ribosomal proteins. To identify enzymes that repair DPCs, we carried out RNA interference screening and found that downregulation of flap endonuclease 1 (FEN1) rendered cells hypersensitive to FA. Since FEN1 possesses 5'-flap endonuclease activity, we hypothesized that FA induces DPC-conjugated 5'-flap DNA fragments that can be processed by FEN1. Indeed, we demonstrate that FA damages DNA bases that are converted into 5'-flap via the base excision pathway (BER). We also observed that the damaged DNA bases were colocalized with DPCs and FEN1. Mechanistically, we showed that FEN1 repairs FA-induced DPCs in vivo and cleaves 5'-flap DNA substrate harboring DPC mimetic in vitro. We also found that FEN1 repairs enzymatic topoisomerase II (TOP2)-DPCs induced by their inhibitors etoposide and doxorubicin independently of the BER pathway, and that FEN1 and the DPC-targeting protease SPRTN act as parallel pathways for the repair of both FA-induced non-enzymatic DPCs and etoposide-induced enzymatic TOP2-DPCs. Notably, we found that FA-induced non-enzymatic DPCs and enzymatic TOP2-DPCs are promptly modified by poly-ADP-ribosylation (PARylation), a post-translational modification catalyzed by PARP1, a key DNA damage response effector that acts by PARylating both DNA damage sites and DNA repair proteins. We performed immunoprecipitation (IP) assays with anti-PAR antibody for HPLC-MS and identified FEN1 as a PARylation substrate. Next, we showed that PARylation of DPC substrates signaled FEN1 whereas PARylation of FEN1 drove FEN1 to DPC sites. Finally, using the enzymatic labeling of the terminal ADP-ribose-MS method, we identified the E285 residue of FEN1 as a dominant PARylation site, which appeared to be required for FEN1 relocation to DPCs. Taken together, our work not only unveiled the identities of FA-induced DPCs but also discovered an unprecedented PARP1-FEN1 nuclease pathway as a universal and imperative mechanism to repair the miscellaneous DPCs and prevent DPC-induced genomic instability.

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Section: Resultsmentioning

confidence: 99%

Flap endonuclease 1 repairs DNA-protein crosslinks via ADP-ribosylation

Sun,

Jenkins,

El Touny

et al. 2023

Preprint

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“…We examined the accumulation of trapped TOP2 using the ICE bioassay (Anand et al, 2018;Nitiss et al, 2021)by transiently transfecting RH30 cells, a pediatric rhabdomyosarcoma cell line with siRNA against MRE11 or non-targeting siRNA (Figure 1A) followed by treatment with etoposide at various concentrations (2, 10, 50 μM) for 2 h. In accord with findings that MRN complex is involved in the release of the TOP2-like protein Spo11 from DNA in S. cerevisiae and that Rad32 (Mre11) plays a role in Top2 removal in S. pombe (Hartsuiker et al, 2009), we found that both hTOP2α and βcc levels in MRE11-deficient cells were distinctly higher than those in WT cells (transfected with control siRNA) treated with 10 μM etoposide (Figure 1B). However, for etoposide treatments at low concentration (2 μM) or high concentration (50 μM), we failed to observe a significant difference in TOP2α or TOP2βcc levels between MRE11 knockdown cells and WT cells.…”

Section: Resultsmentioning

confidence: 99%

“…Other approaches for detecting elevated levels of TOP2ccs and based on similar biophysical principles have been described, including the TARDIS assay (Willmore et al, 1998) and the RADAR assay (Kiianitsa and Maizels, 2013). A detailed description of some of these assays along with detailed methodological considerations has recently been presented (Nitiss et al, 2021). The ICE assay has been previously used to demonstrate the importance of SUMO, ubiquitin and poly (ADP-ribose) modification of topoisomerases in repairing the enzyme-induced damage (Sun , 2020a;Sun et al, 2021;Sun et al, 2022b), potential roles of p53 in the regulation of repair functions for topoisomeraseinduced damage (Menendez et al, 2022), and the identification of novel repair activities (JLN, unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Requirements for MRN endonuclease processing of topoisomerase II-mediated DNA damage in mammalian cells

Sun

Soans

Mishina

et al. 2022

Front. Mol. Biosci.

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During a normal topoisomerase II (TOP2) reaction, the enzyme forms a covalent enzyme DNA intermediate consisting of a 5′ phosphotyrosyl linkage between the enzyme and DNA. While the enzyme typically rejoins the transient breakage after strand passage, a variety of conditions including drugs targeting TOP2 can inhibit DNA resealing, leading to enzyme-mediated DNA damage. A critical aspect of the repair of TOP2-mediated damage is the removal of the TOP2 protein covalently bound to DNA. While proteolysis plays a role in repairing this damage, nucleolytic enzymes must remove the phosphotyrosyl-linked peptide bound to DNA. The MRN complex has been shown to participate in the removal of TOP2 protein from DNA following cellular treatment with TOP2 poisons. In this report we used an optimized ICE (In vivo Complex of Enzyme) assay to measure covalent TOP2/DNA complexes. In agreement with previous independent reports, we find that the absence or inhibition of the MRE11 endonuclease results in elevated levels of both TOP2α and TOP2β covalent complexes. We also examined levels of TOP2 covalent complexes in cells treated with the proteasome inhibitor MG132. Although MRE11 inhibition plus MG132 was not synergistic in etoposide-treated cells, ectopic overexpression of MRE11 resulted in removal of TOP2 even in the presence of MG132. We also found that VCP/p97 inhibition led to elevated TOP2 covalent complexes and prevented the removal of TOP2 covalent complexes by MRE11 overexpression. Our results demonstrate the existence of multiple pathways for proteolytic processing of TOP2 prior to nucleolytic processing, and that MRE11 can process TOP2 covalent complexes even when the proteasome is inhibited. The interactions between VCP/p97 and proteolytic processing of TOP2 covalent complexes merit additional investigation.

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“…The DUST assay ( 8 ) is an extension of the RADAR assay developed by Maizels and colleagues for detecting proteins covalently bound to DNA ( 49 , 50 ). The DUST assay is an extension of the assay that also allows detection of posttranslational modifications on proteins covalently bound to DNA by using antibodies directed against the modifications.…”

Section: Methodsmentioning

confidence: 99%

RAD54L2 counters TOP2-DNA adducts to promote genome stability

D’Alessandro,

Morales-Juarez,

Richards

et al. 2023

Sci. Adv.

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The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.

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Topoisomerase Assays

Cited by 21 publications

References 118 publications

Flap endonuclease 1 repairs DNA-protein crosslinks via ADP-ribosylation

Flap endonuclease 1 repairs DNA-protein crosslinks via ADP-ribosylation

Requirements for MRN endonuclease processing of topoisomerase II-mediated DNA damage in mammalian cells

RAD54L2 counters TOP2-DNA adducts to promote genome stability

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