Biology subjects

Nitiss, J. L.

Publications and source records attributed to Nitiss, J. L..

4 recordsLinked to original sources

bioRxiv2026

A multi-species toolkit of TOP2 hypercleavage mutants for studying topoisomerase II-mediated DNA damage

DNA topoisomerase II (TOP2) generates transient DNA double-strand breaks that are trapped as TOP2-DNA covalent complexes (TOP2cc) by antibiotic and chemotherapy drugs. Here, we characterize tools for study of cellular responses to TOP2cc, exploiting a Saccharomyces cerevisiae TOP2 mutant (TOP2-F1025Y,R1128G) that generates spontaneous and inhibitor-induced covalent complexes at elevated frequencies. This Top2-hc (for "hypercleavage") mutant protein inhibits yeast cell growth when expressed alone or with endogenous Top2, and growth defects are exacerbated in DNA-repair-deficient genetic backgrounds and/or in the presence of low doses of the Top2 poison mAMSA. We generated analogous mutations in human and mouse TOP2A and TOP2B that gave increased TOP2cc, hypersensitization to topoisomerase poisons, increased DNA damage, and decreased cell survival in cultured cells. We further established knock-in mouse models with inducible, tissue-specific expression of each TOP2-hc isoform, demonstrating overt organismal toxicity and cellular markers of DNA damage responses. To illustrate the potential of these genetic tools, we carried out proof-of-principle screens in yeast and cultured human cells for sensitivity to TOP2-hc. The yeast screen revealed strong requirements for homologous recombination, moderate roles for sister chromatid cohesion and kinetochore function, and dependencies on vesicle and vacuolar functions. The pilot shRNA screen in human cells revealed shared requirements for resistance to expression of either TOP2A-hc or TOP2B-hc as well as examples of isoform specificity. These findings establish hypercleavage mutant proteins as effective tools for studying topoisomerase isoform-specific DNA damage and offer a foundation for exploring TOP2cc toxicity and tolerance in vivo. Significance statementDNA topoisomerase II enzymes untangle chromosomes by cutting DNA, but incomplete resealing creates toxic damage that is the basis of antibacterial and chemotherapy drugs. Here we describe toolkits in yeast, mammalian cells, and mice that take advantage of mutant topoisomerase II enzymes that trap on DNA without drugs, creating powerful genetic systems to better study how cells deal with this type of DNA damage. We provide benchmarking data to validate these tools and to illustrate how they can be used for screens in cultured cells or tissue-specific experiments in vivo. These toolkits overcome longstanding technical barriers and enable new ways to study topoisomerase II-mediated DNA damage.

genetics↗

bioRxiv2023

Modeling Allosteric Mechanisms of Eukaryotic Type II Topoisomerases

Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA metabolic processes. These enzymes function as large, homodimeric complexes that undergo a complex cycle of binding and hydrolysis of two ATP molecules in their ATPase domains, which regulates the capture and passage of one DNA double-helix through a second, cleaved DNA molecule. This process requires the transmission of information about the state of the bound nucleotide over vast ranges in the TopoII complex. How this information is transmitted at the molecular level to regulate TopoII functions and how protein substitutions disrupt these mechanisms remains largely unknown. Here, we employed extensive microsecond scale molecular dynamics simulations of the yeast TopoII enzyme in multiple nucleotide-bound states and with amino acid substitutions near both the N- and C-terminals of the complex. Simulation results indicate that the ATPase domains are remarkably flexible on the sub-microsecond timescale and that these dynamics are modulated by the identity of the bound nucleotides and both local and distant amino acid substitutions. Network analyses point towards specific allosteric networks that transmit information about the hydrolysis cycle throughout the complex, which include residues in both the protein and the bound DNA molecule. Amino acid substitutions weaken many of these pathways. Together, our results provide molecular-level details on how the TopoII catalytic cycle is controlled through nucleotide binding and hydrolysis and how mutations may disrupt this process. SIGNIFICANCETypeII Topoisomerases (TopoIIs) are essential and ubiquitous for maintaining DNA topology in the nucleus. The mechanisms by which information about the nucleotide-binding state is transmitted from the ATPase domains throughout the TopoII complex remain poorly understood. We used microsecond timescale molecular dynamics simulations to probe the allosteric mechanisms underlying TopoII function. Results indicate remarkable flexibility of the ATPase domains on this timescale which is modulated by nucleotide binding and local and distant amino acid substitutions. Furthermore, we mapped the allosteric networks linking the ATPase and DNA cleavage domains, and connected them to the ATPase domain dynamics. Our findings provide molecular-level insights into how nucleotide binding and hydrolysis regulate the TopoII catalytic cycle and how mutations can disrupt these processes.

biophysics↗

bioRxiv2023

Using energy to go downhill - a genoprotective role for ATPase activity in DNA topoisomerase II

SO_SCPLOWUMMARYC_SCPLOWType II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction. Curiously, most type II topoisomerases (topos II, IV, and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase II {beta} (hTOP2{beta}) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss leads to increased DNA nicking and double strand break formation by the enzyme. The unstructured C-terminal domains (CTDs) of hTOP2{beta} strongly potentiate strand passage activity in the absence of the ATPase regions, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleavaingevitro, as well as in vivo. By contrast, the aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are restored. Our findings are consistent with the proposal that type II topoisomerases acquired an ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage.

biochemistry↗

bioRxiv2019

A conserved SUMO-Ubiquitin pathway directed by RNF4/SLX5-SLX8 and PIAS4/SIZ1 drives proteasomal degradation of topoisomerase DNA-protein crosslinks

Topoisomerase cleavage complexes (TOPccs) can be stalled physiologically and by the anticancer drugs camptothecins (TOP1 inhibitors) and etoposide (TOP2 inhibitor), yielding irreversible TOP DNA-protein crosslinks (TOP-DPCs). Here we elucidate how TOP-DPCs are degraded via the SUMO-ubiquitin (Ub) pathway. We show that in human cells, TOP-DPCs are promptly and sequentially conjugated by SUMO-2/3, SUMO-1 and Ub. SUMOylation is catalyzed by the SUMO ligase PIAS4, which forms a complex with both TOP1 and TOP2 and {beta}. RNF4 acts as the SUMO-targeted ubiquitin ligase (STUbL) for both TOP1- and TOP2-DPCs in a SUMO-dependent but replication/transcription-independent manner. This SUMO-Ub pathway is conserved in yeast with Siz1 the ortholog of PIAS4 and Slx5-Slx8 the ortholog of RNF4. Our study reveals a conserved SUMO-dependent ubiquitylation pathway for proteasomal degradation of both TOP1- and TOP2-DPCs and potentially for other DPCs.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC=\"FIGDIR/small/707661v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@b00b16org.highwire.dtl.DTLVardef@1a81254org.highwire.dtl.DTLVardef@14b75c1org.highwire.dtl.DTLVardef@12000ba_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefTopoisomerase DNA-protein crosslinks (TOP-DPCs) are the therapeutic mechanism of clinical TOP inhibitors (camptothecin and etoposide). TOP-DPCs induce rapid and sequential conjugation of SUMO-2/3- SUMO-1 and ubiquitin catalyzed by activation of PIAS4 through its DNA-binding SAP domain and RNF4 through its SIM domains. This SUMO-ubiquitin cascade triggers proteasomal degradation of TOP-DPCs.\n\nHIGHLIGHTSO_LIAbortive topoisomerase I (TOP1) and II (TOP2) cleavage complexes resulting in DNA-protein crosslinks (TOP-DPCs) are rapidly and sequentially modified by SUMO-2/3, SUMO-1 and ubiquitin before their proteasomal degradation.\nC_LIO_LIPIAS4 SUMOylates TOP-DPCs via its DNA-binding SAP domain independently of DNA transactions and DNA damage responses.\nC_LIO_LIRNF4 ubiquitylates SUMOylated TOP-DPCs and drives their proteasomal degradation.\nC_LIO_LITOP-DPC processing by the SUMO-Ub pathways is conserved in yeast and human cells.\nC_LI

molecular biology↗