Human DNA topoisomerase I (Topo I) is an essential enzyme in regulating DNA supercoiling during transcription and replication, and it is an important therapeutic target for anti-tumor agents. Bidens pilosa L. is a medicinal herb that is used as a folk medicine for cancers in China. A new flavonoid (1) and a new polyacetylene (20), along with eighteen flavonoids (2-19) and nine polyacetylenes (21-29), were isolated and identified from the methanol extract of the whole plant of B. pilosa, and some of the compounds (4, 5, 6 and 7) exhibited potent cytotoxicity against a panel of five human cancer cell lines. The DNA relaxation assay revealed that some flavonoids and polyacetylenes exerted inhibitory activities on human DNA Topo I, among them compounds 1, 2, 5, 6, 7, 8, 15, 19, 20, 22, and 24 were the most active ones, with IC50 values of 393.5, 328.98, 145.57, 239.27, 224.38, 189.84, 89.91, 47.5, 301.32, 178.03, and 218.27 μM, respectively. The structure-activity analysis of flavonoids was performed according to the results from the Topo I inhibition assay. The DNA content analysis revealed that 5, 6, and 7 potently arrested cell cycle at the G1/S and G2/M phases in human colon cancer cell DLD-1 depending on the concentration of the inhibitors. The levels of protein expression related to the G1/S and G2/M cell cycle checkpoints were in accordance with the results from the DNA content analysis. These findings suggest that flavonoids are one of the key active ingredients accounting for the anti-tumor effect of B. pilosa.

DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors.

Agarose gel electrophoresis in the presence of chloroquine (an intercalating agent) can be used to resolve and characterize the population of topoisomers present in supercoiled plasmid DNA. Here, we describe how chloroquine gel electrophoresis can capture changes in the topoisomer distribution of plasmid DNA that bears a recognition site for a given protein, if that plasmid is isolated from cells producing the protein of interest. We also describe two complementary in vitro assays, which can be used to capture transient changes in DNA supercoiling caused when the purified protein of interest engages its recognition site. These are the topoisomerase I-mediated relaxation assay (TMRA) and the ligase-mediated supercoiling assay (LMSA). Together, these in vivo and in vitro methods allow the capture and measurement of changes in DNA topology that are triggered by DNA-binding proteins, especially those that multimerize on or spread along DNA.

Human topoisomerase III beta (hTOP3B) is the only topoisomerase in the human cell that can act on both DNA and RNA substrates. Recent findings have emphasized the physiological importance of hTOP3B and consolidated it as a valuable drug target for antiviral and anticancer therapeutics. Although type IA topoisomerases of different organisms have been studied over the years, the step-by-step interaction of hTOP3B and nucleic acid substrates is still not well understood. Due to the lack of hTOP3B-RNA structures as well as DNA/RNA covalent complexes, computational investigations have been limited. In our study, we utilized molecular dynamics (MD) simulations to study the interactions between hTOP3B and nucleic acids to get a closer look into the residues that play a role in binding DNA or RNA and facilitate catalysis, along with the differences and similarities when hTOP3B interacts with DNA compared to RNA. For this, we generated multiple models of hTOP3B complexed with DNA and RNA sequences using the hTOP3B crystal structure and 8-mer single-stranded DNA and RNA sequences. These models include both covalent and noncovalent complexes, which are then subjected to MD simulations and analyzed. Our findings highlight the complexes\' stability, sequence preference, and interactions of the binding pocket residues with different nucleotides. Our work demonstrates that hTOP3B forms stable complexes with both DNA and RNA and provides a better understanding of the enzyme\'s interaction with different nucleic acid substrate sequences.

Downstream-of-gene (DoG) transcripts are an emerging class of noncoding RNAs. However, it remains largely unknown how DoG RNA production is regulated and whether alterations in DoG RNA signatures exist in major cancers. Here, through transcriptomic analyses of matched tumors and nonneoplastic tissues and cancer cell lines, we reveal a comprehensive catalog of DoG RNA signatures. Through separate lines of evidence, we support the biological importance of DoG RNAs in carcinogenesis. First, we show tissue-specific and stage-specific differential expression of DoG RNAs in tumors versus paired normal tissues with their respective host genes involved in tumor-promoting versus tumor-suppressor pathways. Second, we identify that differential DoG RNA expression is associated with poor patient survival. Third, we identify that DoG RNA induction is a consequence of treating colon cancer cells with the topoisomerase I (TOP1) poison camptothecin and following TOP1 depletion. Our results underlie the significance of DoG RNAs and TOP1-dependent regulation of DoG RNAs in diversifying and modulating the cancer transcriptome.

RNA Polymerase (RNAP) II transcription on non-coding repetitive satellite DNAs plays an important role in chromosome segregation, but a little is known about the regulation of satellite transcription. We here show that Topoisomerase I (TopI), not TopII, promotes the transcription of α-satellite DNAs, the main type of satellite DNAs on human centromeres. Mechanistically, TopI localizes to centromeres, binds RNAP II and facilitates RNAP II elongation. Interestingly, in response to DNA double-stranded breaks (DSBs), α-satellite transcription is dramatically stimulated in a DNA damage checkpoint-independent but TopI-dependent manner, and these DSB-induced α-satellite RNAs form into strong speckles in the nucleus. Remarkably, TopI-dependent satellite transcription also exists in mouse 3T3 and Drosophila S2 cells and in Drosophila larval imaginal wing discs and tumor tissues. Altogether, our findings herein reveal an evolutionally conserved mechanism with TopI as a key player for the regulation of satellite transcription at both cellular and animal levels.

All cells must maintain the structural and functional integrity of the genome under a wide range of environments. High temperatures pose a formidable challenge to cells by denaturing the DNA double helix, causing chemical damage to DNA, and increasing the random thermal motion of chromosomes. Thermophiles, predominantly classified as bacteria or archaea, exhibit an exceptional capacity to mitigate these detrimental effects and prosper under extreme thermal conditions, with some species tolerating temperatures higher than 100°C. Their genomes are mainly characterized by the presence of reverse gyrase, a unique topoisomerase that introduces positive supercoils into DNA. This enzyme has been suggested to maintain the genome integrity of thermophiles by limiting DNA melting and mediating DNA repair. Previous studies provided significant insights into the mechanisms by which NAPs, histones, SMC superfamily proteins, and polyamines affect the 3D genomes of thermophiles across different scales. Here, I discuss current knowledge of the genome organization in thermophiles and pertinent research questions for future investigations.

DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage complex (Top1cc) to allow strand rotation and duplex relaxation, which can lead to elevated levels of DNA-RNA hybrids and micronuclei. To comprehend the underlying mechanisms, we have integrated genomic data of Top1cc-triggered hybrids and DNA double-strand breaks (DSBs) shortly after Top1cc induction, revealing that Top1ccs increase hybrid levels with different mechanisms. DSBs are at highly transcribed genes in early replicating initiation zones and overlap with hybrids downstream of accumulated RNA polymerase II (RNAPII) at gene 5\'-ends. A transcription factor IIS mutant impairing transcription elongation further increased RNAPII accumulation likely due to backtracking. Moreover, Top1ccs can trigger micronuclei when occurring during late G1 or early/mid S, but not during late S. As micronuclei and transcription-replication conflicts are attenuated by transcription factor IIS, our results support a role of RNAPII arrest in Top1cc-induced transcription-replication conflicts leading to DSBs and micronuclei.

Inhibitors of a DNA repair enzyme known as polynucleotide kinase 3\'-phosphatase (PNKP) are expected to show synergistic cytotoxicity in combination with topoisomerase I (TOP1) inhibitors in cancer. In this study, the synergistic cytotoxicity of a novel inhibitor of PNKP, i.e., A83B4C63, with a potent TOP1 inhibitor, i.e., SN-38, against colorectal cancer cells was investigated. Polymeric micelles (PMs) for preferred tumor delivery of A83B4C63, developed through physical encapsulation of this compound in methoxy poly(ethylene oxide)-poly(α-benzyl carboxylate-ε-caprolactone) (mPEO-b-PBCL) micelles, were combined with SN-38 in free or PM form. The PM form of SN-38 was prepared through chemical conjugation of SN-38 to the functional end group of mPEO-b-PBCL and further assembly of mPEO-b-PBCL-SN-38 in water. Moreover, mixed micelles composed of mPEO-b-PBCL and mPEO-b-PBCL-SN-38 were used to co-load A83B4C63 and SN-38 in the same nanoformulation. The loading content (% w/w) of the SN-38 and A83B4C63 to mPEO-b-PBCL in the co-loaded formulation was 7.91 ± 0.66 and 16.13 ± 0.11% (w/w), respectively, compared to 15.67 ± 0.34 (% w/w) and 23.06 ± 0.63 (% w/w) for mPEO-b-PBCL micelles loading individual drugs. Notably, the average diameter of PMs co-encapsulating both SN-38 and A83B4C63 was larger than that of PMs encapsulating either of these compounds alone but still lower than 60 nm. The release of A83B4C63 from PMs co-encapsulating both drugs was 76.36 ± 1.41% within 24 h, which was significantly higher than that of A83B4C63-encapsulated micelles (42.70 ± 0.72%). In contrast, the release of SN-38 from PMs co-encapsulating both drugs was 44.15 ± 2.61% at 24 h, which was significantly lower than that of SN-38-conjugated PMs (74.16 ± 3.65%). Cytotoxicity evaluations by the MTS assay as analyzed by the Combenefit software suggested a clear synergy between PM/A83B4C63 (at a concentration range of 10-40 μM) and free SN-38 (at a concentration range of 0.001-1 μM). The synergistic cytotoxic concentration range for SN-38 was narrowed down to 0.1-1 or 0.01-1 μM when combined with PM/A83B4C63 at 10 or 20-40 μM, respectively. In general, PMs co-encapsulating A83B4C63 and SN-38 at drug concentrations within the synergistic range (10 μM for A83B4C63 and 0.05-1 μM for SN-38) showed slightly less enhancement of SN-38 anticancer activity than a combination of individual micelles, i.e., A83B4C63 PMs + SN-38 PMs at the same molar concentrations. This was attributed to the slower release of SN-38 from the SN-38 and A83B4C63 co-encapsulated PMs compared to PMs only encapsulating SN-38. Cotreatment of cells with TOP1 inhibitors and A83B4C63 formulation enhanced the expression level of γ-HA2X, cleaved PARP, caspase-3, and caspase-7 in most cases. This trend was more consistent and notable for PMs co-encapsulating both A83B4C63 and SN-38. The overall result from the study shows a synergy between PMs of SN-38 and A83B4C63 as a mixture of two PMs for individual drugs or PMs co-encapsulating both drugs.

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.