Human DNA topoisomerase I poisoning causes R loop-mediated genome instability attenuated by transcription factor IIS

With our study we propose a role of RNAPII arrest in Top1cc-induced transcription-replication conflicts, leading to DSBs, micronuclei and genome instability.
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DNA topoisomerases are a family of essential nuclear enzymes that regulate DNA topology, chromatin structures, and basic processes such as transcription and replication. DNA topoisomerase I (Top1) couples DNA strand cleavage-ligation reaction with the rotation of the cut strand around the other, therefore reducing the torsional tension of DNA duplexes. During the catalytic cycle, a reaction intermediate called Top1-DNA cleavage complex (Top1cc) forms, wherein Top1 is covalently linked to the 3′-end of the cut strand. However, under certain circumstances, Top1ccs can trigger genome instability. Top1ccs can be stabilized by several factors, including U.S. Food and Drug Administration–approved antitumor Top1 poisons, such as camptothecin (CPT) analogs. CPT selectively targets Top1 in living cells, leading to a rapid (2 to 5 min) increase in Top1ccs. Within minutes, they affect RNA and DNA synthesis markedly, impair DNA transactions and epigenomic features, and activate ubiquitin-dependent Top1 degradation. Moreover, CPT-induced Top1ccs increase transcription-replication conflicts (TRCs) and DNA double-strand breaks (DSBs). The generation of DSBs may be due to replication run off at Top1cc sites or to the activity of endonucleases at stalled forks. TRCs occur more often in cancer than normal cells due to high oncogene-induced transcription rates and can thus be at the basis of high genome instability in cancer.

Top1ccs can also occur in unperturbed cells as they can arise when the DNA substrate is damaged. Top1ccs were shown to be a general pathogenic feature of neurodegenerative disorders derived from mutations of DNA repair factors. A mutation of Tyrosyl-DNA phosphodiesterase 1 (TDP1), a specific repair factor of Top1ccs, can cause a rare type of ataxia spinocerebellar ataxia with axonal neuropathy 1 (SCAN1) characterized by cell hypersensitivity to CPT. Moreover, Top1 can introduce short indels in the genome at sites of misincorporated ribonucleotides. Thus, impaired Top1 activity can be dangerous to cell life and differentiation when nuclear Top1cc levels become elevated because of exogenous or endogenous factors. However, the mechanism elicited by Top1ccs is not yet fully understood.

Top1ccs can effectively induce TRCs with a yet undefined R loop–mediated mechanism. R loops are three-strand nucleic acid structures containing a hybrid DNA:RNA duplex. Top1 poisons cause a transient increase in R loops in cancer cells. However, genomic R loop maps are not yet available upon Top1cc increase. Recently, we showed that micronuclei induced by Top1ccs are dependent on R loops. In addition, mutations of ribonuclease (RNase) H2, an endonuclease specifically targeting RNA strands of hybrid duplexes, can cause the human Aicardi-Goutières syndrome and an increase in micronuclei triggering a persistent inflammatory activation, likely at the basis of the syndrome. However, the mechanism of hybrid-dependent TRCs, DNA cleavage, and micronucleus formation remains largely unknown in human cancer cells.

In this study, we uncover a main mechanism by which Top1ccs induce R loops and TRCs. By determining the genomic maps of DNA-RNA hybrid and DSB loci upon Top1cc induction, we provide evidence for the role of transcription elongation factor IIS (TFIIS) in attenuating TRCs and genome instability. Moreover, analysis of cell cycle phase–dependent triggering of micronuclei allowed us to show that Top1ccs affect genome stability specifically during late G1/early S phases. Our findings establish an early replication-specific and transcription-dependent mechanism of genome instability elicited by Top1ccs and provide the main genomic sites of transcription/replication conflicts (TCRs) induced by Top1ccs. These findings can aid in the development of strategies for neurodegeneration prevention and cancer treatment.

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DNA Damage and Repair
Life Sciences > Biological Sciences > Molecular Biology > DNA Damage and Repair
Genomic Instability
Life Sciences > Biological Sciences > Genetics and Genomics > Molecular Genetics > Genomic Instability