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Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

National Institutes of Health

Topoisomerases introduce transient DNA breaks to relax supercoiled DNA, remove catenanes and enable chromosome segregation. Human cells encode six topoisomerases (TOP1, TOP1mt, TOP2α, TOP2β, TOP3α and TOP3β), which act on a broad range of DNA and RNA substrates at the nuclear and mitochondrial genomes. Their catalytic intermediates, the topoisomerase cleavage complexes (TOPcc), are therapeutic targets of various anticancer drugs. TOPcc can also form on damaged DNA during replication and transcription, and engage specific repair pathways, such as those mediated by tyrosyl-DNA phosphodiesterase 1 (TDP1) and TDP2 and by endonucleases (MRE11, XPF-ERCC1 and MUS81). Here, we review the roles of topoisomerases in mediating chromatin dynamics, transcription, replication, DNA damage repair and genomic stability, and discuss how deregulation of topoisomerases can cause neurodegenerative diseases, immune disorders and cancer.

Roles of eukaryotic topoisomerases in transcription, replication and genomic stability.

Topoisomerase II (TOP2) relieves topological stress in DNA by introducing double-strand breaks (DSBs) via a transient, covalently linked TOP2 DNA-protein intermediate, termed TOP2 cleavage complex (TOP2cc). TOP2ccs are normally rapidly reversible, but can be stabilized by TOP2 poisons, such as the chemotherapeutic agent etoposide (ETO). TOP2 poisons have shown significant variability in their therapeutic effectiveness across different cancers for reasons that remain to be determined. One potential explanation for the differential cellular response to these drugs is in the manner by which cells process TOP2ccs. Cells are thought to remove TOP2ccs primarily by proteolytic degradation followed by DNA DSB repair. Here, we show that proteasome-mediated repair of TOP2cc is highly error-prone. Pre-treating primary splenic mouse B-cells with proteasome inhibitors prevented the proteolytic processing of trapped TOP2ccs, suppressed the DNA damage response (DDR) and completely protected cells from ETO-induced genome instability, thereby preserving cellular viability. When degradation of TOP2cc was suppressed, the TOP2 enzyme uncoupled itself from the DNA following ETO washout, in an error-free manner. This suggests a potential mechanism of developing resistance to topoisomerase poisons by ensuring rapid TOP2cc reversal.

Suppressing proteasome mediated processing of topoisomerase II DNA-protein complexes preserves genome integrity.

Topoisomerases are essential enzymes solving DNA topological problems such as supercoils, knots and catenanes that arise from replication, transcription, chromatin remodeling and other nucleic acid metabolic processes. They are also the targets of widely used anticancer drugs (e.g. topotecan, irinotecan, enhertu, etoposide, doxorubicin, mitoxantrone) and fluoroquinolone antibiotics (e.g. ciprofloxacin and levofloxacin). Topoisomerases manipulate DNA topology by cleaving one DNA strand (TOP1 and TOP3 enzymes) or both in concert (TOP2 enzymes) through the formation of transient enzyme-DNA cleavage complexes (TOPcc) with phosphotyrosyl linkages between DNA ends and the catalytic tyrosyl residue of the enzymes. Failure in the self-resealing of TOPcc results in persistent TOPcc (which we refer it to as topoisomerase DNA-protein crosslinks (TOP-DPC)) that threaten genome integrity and lead to cancers and neurodegenerative diseases. The cell prevents the accumulation of topoisomerase-mediated DNA damage by excising TOP-DPC and ligating the associated breaks using multiple pathways conserved in eukaryotes. Tyrosyl-DNA phosphodiesterases (TDP1 and TDP2) cleave the tyrosyl-DNA bonds whereas structure-specific endonucleases such as Mre11 and XPF (Rad1) incise the DNA phosphodiester backbone to remove the TOP-DPC along with the adjacent DNA segment. The proteasome and metalloproteases of the WSS1/Spartan family typify proteolytic repair pathways that debulk TOP-DPC to make the peptide-DNA bonds accessible to the TDPs and endonucleases. The purpose of this review is to summarize our current understanding of how the cell excises TOP-DPC and why, when and where the cell recruits one specific mechanism for repairing topoisomerase-mediated DNA damage, acquiring resistance to therapeutic topoisomerase inhibitors and avoiding genomic instability, cancers and neurodegenerative diseases.

Excision repair of topoisomerase DNA-protein crosslinks (TOP-DPC).

Topoisomerases form transient covalent DNA cleavage complexes to perform their reactions. Topoisomerase I cleavage complexes (TOP1ccs) are trapped by camptothecin and TOP2ccs by etoposide. Proteolysis of the trapped topoisomerase DNA-protein cross-links (TOP-DPCs) is a key step for some pathways to repair these lesions. We describe a pathway that features a prominent role of the small ubiquitin-like modifier (SUMO) modification for both TOP1- and TOP2-DPC repair. Both undergo rapid and sequential SUMO-2/3 and SUMO-1 modifications in human cells. The SUMO ligase PIAS4 is required for these modifications. RNF4, a SUMO-targeted ubiquitin ligase (STUbL), then ubiquitylates the TOP-DPCs for their subsequent degradation by the proteasome. This pathway is conserved in yeast with Siz1 and Slx5-Slx8, the orthologs of human PIAS4 and RNF4.

A conserved SUMO pathway repairs topoisomerase DNA-protein cross-links by engaging ubiquitin-mediated proteasomal degradation.

We demonstrate diffraction-limited and super-resolution imaging through thick layers (tens-hundreds of microns) of BIO-133, a biocompatible, UV-curable, commercially available polymer with a refractive index (RI) matched to water. We show that cells can be directly grown on BIO-133 substrates without the need for surface passivation and use this capability to perform extended time-lapse volumetric imaging of cellular dynamics 1) at isotropic resolution using dual-view light-sheet microscopy, and 2) at super-resolution using instant structured illumination microscopy. BIO-133 also enables immobilization of 1) Drosophila tissue, allowing us to track membrane puncta in pioneer neurons, and 2) Caenorhabditis elegans, which allows us to image and inspect fine neural structure and to track pan-neuronal calcium activity over hundreds of volumes. Finally, BIO-133 is compatible with other microfluidic materials, enabling optical and chemical perturbation of immobilized samples, as we demonstrate by performing drug and optogenetic stimulation on cells and C. elegans.

A polymer index-matched to water enables diverse applications in fluorescence microscopy.

Epigenetic regulators can modulate the effects of cancer therapeutics. To further these observations, we discovered that the bromodomain PHD finger transcription factor subunit (BPTF) of the nucleosome remodeling factor (NURF) promotes resistance to doxorubicin, etoposide, and paclitaxel in the 4T1 breast tumor cell line. BPTF functions in promoting resistance to doxorubicin and etoposide, but not paclitaxel, and may be selective to cancer cells, as a similar effect was not observed in embryonic stem cells. Sensitization to doxorubicin and etoposide with BPTF knockdown (KD) was associated with increased DNA damage, topoisomerase II (TOP2) crosslinking and autophagy; however, there was only a modest increase in apoptosis and no increase in senescence. Sensitization to doxorubicin was confirmed  with the syngeneic 4T1 breast tumor model using both genetic and pharmacologic inhibition of BPTF. The effects of BPTF inhibition  are autophagy dependent, based on genetic autophagy inhibition. Finally, treatment of 4T1, 66cl4, 4T07, MDA-MB-231, but not ER-positive 67NR and MCF7 breast cancer cells with the selective BPTF bromodomain inhibitor, AU1, recapitulates genetic BPTF inhibition, including  sensitization to doxorubicin, increased TOP2-DNA crosslinks and DNA damage. Taken together, these studies demonstrate that BPTF provides resistance to the antitumor activity of TOP2 poisons, preventing the resolution of TOP2 crosslinking and associated autophagy. These studies suggest that BPTF can be targeted with small-molecule inhibitors to enhance the effectiveness of TOP2-targeted cancer chemotherapeutic drugs. IMPLICATIONS: These studies suggest NURF can be inhibited pharmacologically as a viable strategy to improve chemotherapy effectiveness.

Autophagy-Dependent Sensitization of Triple-Negative Breast Cancer Models to Topoisomerase II Poisons by Inhibition of the Nucleosome Remodeling Factor.

Small cell neuroendocrine cancers (SCNCs) are recalcitrant cancers arising from diverse primary sites that lack effective treatments. Using chemical genetic screens, we identified inhibition of ataxia telangiectasia and rad3 related (ATR), the primary activator of the replication stress response, and topoisomerase I (TOP1), nuclear enzyme that suppresses genomic instability, as synergistically cytotoxic in small cell lung cancer (SCLC). In a proof-of-concept study, we combined M6620 (berzosertib), first-in-class ATR inhibitor, and TOP1 inhibitor topotecan in patients with relapsed SCNCs. Objective response rate among patients with SCLC was 36% (9/25), achieving the primary efficacy endpoint. Durable tumor regressions were observed in patients with platinum-resistant SCNCs, typically fatal within weeks of recurrence. SCNCs with high neuroendocrine differentiation, characterized by enhanced replication stress, were more likely to respond. These findings highlight replication stress as a potentially transformative vulnerability of SCNCs, paving the way for rational patient selection in these cancers, now treated as a single disease.

Therapeutic targeting of ATR yields durable regressions in small cell lung cancers with high replication stress.

Poly(ADP)-ribosylation (PARylation) regulates chromatin structure and recruits DNA repair proteins. Using single-molecule fluorescence microscopy to track topoisomerase I (TOP1) in live cells, we found that sustained PARylation blocked the repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) in a similar fashion as inhibition of the ubiquitin-proteasome system (UPS). PARylation of TOP1-DPC was readily revealed by inhibiting poly(ADP-ribose) glycohydrolase (PARG), indicating the otherwise transient and reversible PARylation of the DPCs. As the UPS is a key repair mechanism for TOP1-DPCs, we investigated the impact of TOP1-DPC PARylation on the proteasome and found that the proteasome is unable to associate with and digest PARylated TOP1-DPCs. In addition, PARylation recruits the deubiquitylating enzyme USP7 to reverse the ubiquitylation of PARylated TOP1-DPCs. Our work identifies PARG as repair factor for TOP1-DPCs by enabling the proteasomal digestion of TOP1-DPCs. It also suggests the potential regulatory role of PARylation for the repair of a broad range of DPCs.

PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation.

Topoisomerase 2 (TOP2) inhibitors are drugs widely used in the treatment of different types of cancer. Processing of their induced-lesions create double-strand breaks (DSBs) in the DNA, which is the main toxic mechanism of topoisomerase inhibitors to kill cancer cells. It was established that the Nucleotide Excision Repair pathway respond to TOP2-induced lesions, mainly through the Cockayne Syndrome B (CSB) protein. In this paper, we further define the mechanism and type of lesions induced by TOP2 inhibitors when CSB is abrogated. In the absence of TOP2, but not during pharmacological inhibition, an increase in R-Loops was detected. We also observed that CSB knockdown provokes the accumulation of DSBs induced by TOP2 inhibitors. Consistent with a functional interplay, interaction between CSB and TOP2 occurred after TOP2 inhibition. This was corroborated with  DNA cleavage assays where CSB stimulated the activity of TOP2. Altogether, our results show that TOP2 is stimulated by the CSB protein and prevents the accumulation of R-loops/DSBs linked to genomic instability.

Functions of the CSB Protein at Topoisomerase 2 Inhibitors-Induced DNA Lesions.

Confocal microscopy remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.

Multiview confocal super-resolution microscopy.

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 ( 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.

Yilun Sun

Developmental Therapeutics Branch,
Center for Cancer Research,
National Cancer Institute,
Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute

National Institutes of Health

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

Yilun Sun

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Developmental Therapeutics Branch,Center for Cancer Research,National Cancer Institute,Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute

National Institutes of Health

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

Developmental Therapeutics Branch,
Center for Cancer Research,
National Cancer Institute,
Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute