Supercoiling in DNA and chromatin

“The boundaries of TADS are enriched for CTCF binding sites.”

“The boundaries of TADS are enriched for CTCF binding sites.”

“The classic example is the ‘twin supercoiled domain’ model where elongating RNA polymerase, in unwinding the DNA, generates positive supercoiling ahead and, in rewinding the DNA, generates negative supercoiling in its wake [7,8] (Figure 1).”

“The genome is also separately organised into about 500 independent 10 kb supercoiling domains with demarcating barriers stochastically distributed and dynamically maintained [19,20].”

“Genes that are negatively supercoiled are generally more efficiently transcribed [9,10] but topoisomerase inhibition studies [11,12 ,13,14] indicate that the accumulation of excessive positive or negative supercoiling will repress transcription.”

“Although in vitro studies support this concept [5] its operation in vivo is elusive [6].”

“Condensins introduce global positive writhe into the DNA/chromatin in vitro [46] and as a result changes in supercoiling energy are thought to co-dependently drive mitotic chromosome architecture [47] and resolution in vivo.”

“To understand the nature of eukaryotic supercoiling domains, psoralen binding has been used in combination with microarrays to map the distribution of DNA supercoils across entire genomes [23] or to particular chromosomal regions [24 ,25 ].”

“[34 ], in a single-molecule approach, used an angular optical trap.”

“[33] used a tamoxifen-inducible Cre recombinase to excise a chromatin segment with its torsional stress trapped intact.”

“Genes that are negatively supercoiled are generally more efficiently transcribed [9,10] but topoisomerase inhibition studies [11,12 ,13,14] indicate that the accumulation of excessive positive or negative supercoiling will repress transcription.”

“The classic example is the ‘twin supercoiled domain’ model where elongating RNA polymerase, in unwinding the DNA, generates positive supercoiling ahead and, in rewinding the DNA, generates negative supercoiling in its wake [7,8] (Figure 1).”

“Genes that are negatively supercoiled are generally more efficiently transcribed [9,10] but topoisomerase inhibition studies [11,12 ,13,14] indicate that the accumulation of excessive positive or negative supercoiling will repress transcription.”

“On approaching mitosis the remaining catenations, or sister chromatid intertwinings are ‘identified’ by a process that involves an architectural change in chromatin structure, orchestrated by condensin-generated and mitotic spindle-dependant positive supercoiling [44].”

“The capacity of negative supercoiling to unwind DNA and facilitate processes such as transcription [29,30] and replication and its ability to induce alternative DNA structures such as cruciform [31], G-quadruplexes and Z-DNA [32] have been noted.”

“Condensins introduce global positive writhe into the DNA/chromatin in vitro [46] and as a result changes in supercoiling energy are thought to co-dependently drive mitotic chromosome architecture [47] and resolution in vivo.”

“Although we raised this issue, twenty-five years ago [38], the question essentially remains unanswered as the difficulty is multifaceted.”

“A build up of negative supercoiling propagates along the fibre, melting the FUSE element promoting the binding of the ssDNA binding proteins, FBP and FIR [33,48].”

“[3] with the Ter domain being subdivided into smaller, 35 kb domains via MatS/MatP interactions [18].”

“At the level of the ‘twin supercoil domain’ the scenario seems simplistic; positive supercoiling ahead of the polymerase will destabilize nucleosome structure and negative supercoiling behind will promote reassembly [36], actions that seem entirely consistent with the thermodynamic demands of transcription through a c…”

“[42] observed that inhibition of topoisomerase I and the build up of positive supercoiling caused replication delay in long but not short yeast chromosomes.”

“The capacity of negative supercoiling to unwind DNA and facilitate processes such as transcription [29,30] and replication and its ability to induce alternative DNA structures such as cruciform [31], G-quadruplexes and Z-DNA [32] have been noted.”

“Therefore, there must be a regulated balance in the localised levels of supercoiling through the concerted actions of polymerases [15] and topoisomerases [16,17].”

“Chromatin–chromatin interactions may act as supercoil diffusion barriers but the inherent drag, and therefore reduced rotation, caused by higher levels of chromatin organisation could in itself be sufficient to form the basis of supercoiling domains [26,27].”

“The capacity of negative supercoiling to unwind DNA and facilitate processes such as transcription [29,30] and replication and its ability to induce alternative DNA structures such as cruciform [31], G-quadruplexes and Z-DNA [32] have been noted.”

“Therefore, there must be a regulated balance in the localised levels of supercoiling through the concerted actions of polymerases [15] and topoisomerases [16,17].”

“Genes that are negatively supercoiled are generally more efficiently transcribed [9,10] but topoisomerase inhibition studies [11,12 ,13,14] indicate that the accumulation of excessive positive or negative supercoiling will repress transcription.”

“[3] with the Ter domain being subdivided into smaller, 35 kb domains via MatS/MatP interactions [18].”

“Concomitantly, chromosome compaction starts during S-phase when condensin II is recruited to replicated regions [45].”

“If these are not efficiently removed the residual energy may influence DNA or chromatin structure locally [28], or, if the energy can be propagated along the fibre, at more distant sites.”

“Genes that are negatively supercoiled are generally more efficiently transcribed [9,10] but topoisomerase inhibition studies [11,12 ,13,14] indicate that the accumulation of excessive positive or negative supercoiling will repress transcription.”

“To understand the nature of eukaryotic supercoiling domains, psoralen binding has been used in combination with microarrays to map the distribution of DNA supercoils across entire genomes [23] or to particular chromosomal regions [24 ,25 ].”

“The genome is also separately organised into about 500 independent 10 kb supercoiling domains with demarcating barriers stochastically distributed and dynamically maintained [19,20].”

“Genes that are negatively supercoiled are generally more efficiently transcribed [9,10] but topoisomerase inhibition studies [11,12 ,13,14] indicate that the accumulation of excessive positive or negative supercoiling will repress transcription.”

“The capacity of negative supercoiling to unwind DNA and facilitate processes such as transcription [29,30] and replication and its ability to induce alternative DNA structures such as cruciform [31], G-quadruplexes and Z-DNA [32] have been noted.”

“Nucleosomes containing yeast histones are more sensitive to thermally induced torsional stress [39] than nucleosomes containing higher eukaryotic core histones suggesting, perhaps, a greater propensity for yeast chromatin to absorb rather than transmit negative supercoiling.”

“(b) Negative supercoiling promotes ssDNA patch formation allowing the mutagenic activity of activation-induced cytidine deaminase (AID) [35 ].”

“Although in vitro studies support this concept [5] its operation in vivo is elusive [6].”

“Although there is no direct evidence for the transmission of torsion through chromatin the phenomenon is used to explain the apparent dissipation of positive supercoiling near theendsofyeastchromosomes[41].”

“Therefore, there must be a regulated balance in the localised levels of supercoiling through the concerted actions of polymerases [15] and topoisomerases [16,17].”

“However, the many models that purport to explain the mechanics of how polymerase does in fact transcribe through a nucleosome reflects our ignorance of the details [37].”

“Chromatin–chromatin interactions may act as supercoil diffusion barriers but the inherent drag, and therefore reduced rotation, caused by higher levels of chromatin organisation could in itself be sufficient to form the basis of supercoiling domains [26,27].”

“However, when converging forks approach, relaxation of positive supercoiling is restricted and the build up of torsional stress causes swivelling of the replication complex required to complete replication [43 ].”

“If loops are preformed into left-handed (or right-handed) inter-wound supercoils (53), pyramids are more sharply defined (Figure 3E; these results are also consistent with those obtained in (21,33)).”

“Together, these results suggested that the difference in cleavage activity between PCR amplicons and endogenous intracellular loci might be related to various effects of the intracellular condition, such as degradation issues by DNA exonuclease (29), structural hindrances by binding of histones or other proteins (30),…”

“In eukaryotes, DNA replication, transcription, and chromatin-remodeling transiently introduce negative superhelicity (54, 55).”

“Because overtwisted DNA inhibits strand melting and undertwisted DNA promotes it, DNA supercoiling can profoundly impact the binding of regulatory proteins, promoter firing dynamics, DNA replication, and chromosome architecture (Dillon and Dorman, 2010; Gilbert and Allan, 2014).”

“This strand displacement would be facilitated by negative supercoiling, which is present (either transiently or permanently) in many genomes (reviewed in (40,41)).”

“Negative superhelicity is also thought to facilitate transcription elongation, whereas excessive positive supercoiling impedes it (28).”

“and elongation steps, and can thereby be considered as a non-conventional transcriptional regulator in eukaryotes as well as bacteria [1, 2, 3, 4].”

“But it also depends crucially on the presence of torsional stress [4] giving rise to supercoiling, a mechanical feature present in virtually all biological transactions involving DNA [5,6], and in particular transcription and replication.”

“Biochemically, DNA TOPs cleave the phosphodiester bond in a coordinated way and religate the ends of DNA to unwind or wind DNA to control the supercoiling (53) and resolve disordered DNA entanglements and knots (54, 55).”

“A promising method involving the intercalating agent psoralen was developed in eukaryotes [7,102] but did not yet provide high-resolution data in bacteria [1].”

“In principle, when positive supercoils generated during transcription are preferentially relaxed by DNA topoisomerases, transcription is able to introduce one negative supercoil for every 10 nucleotides that are transcribed (75).”

“…genomic DNA is torsionally constrained by cellular structures and architectural nucleoproteins (nucleosomes, NAPs, other DNA-binding proteins) ( 4 , 8 , 13 , 16 , 63 ), the translocation of RNAP during transcription causes over-winding of the DNA downstream from the RNAP, and compensatory…”

“As a result, negative supercoiling improves transcription elongation, while positive supercoiling inhibits it (Gilbert and Allan, 2014).”

“Elongating polymerases generate forces and torques (leading to supercoiling [31,32]) which may affect cohesin diffusion; indeed recent experiments where the WAPL cohesin unloader protein is knocked down in mouse nuclei show that cohesin collects preferentially between convergent genes, indicating that polymerase can p…”

“Large-scale folding of chromatin by condensin-mediated positive writhing increases torsional stress in the DNA (Gilbert and Allan 2014).”

“…we propose that persistent levels of ( )TS are achieved via the unbalanced relaxation of (+) and ( )TS. Chromatin architecture and its epigenetic modifications are likely to determine how DNA torsional energy is buffered, dissipated or confined within topological domains (Gilbert & Allan, 2014).”

“proteins, such as replication, chromatin remodeling, and especially transcription; RNA polymerase, for example, introduces approximately seven supercoils per second.(1) As excessive supercoiling can impede transcription or potentiate energy that alters the DNA structure at inappropriate times,(1,2) the relaxation of s…”

“Positively supercoiled (+SC) DNA accumulates in every organism during DNA transactions, such as replication and transcription [15]; moreover, +SC DNA prevents extensive reversal of replication forks in the presence of replication blocking lesions [16], promotes telomere resolutions [17] and marks centromeres as unique…”

“During mitosis, inter-chromosomal, but not intra-chromosomal, crossovers are thought to be identified and subsequently removed by Topoisomerase II (for a review see [17]).”

“axial rotation that would relieve supercoiling (Duncan et al. 1994; Ner et al. 1994; Yang et al. 1995; Gilbert and Allan 2014).”

“”(6) During transcription, it is estimated that one positive supercoil and one negative supercoil are generated for every 10 transcribed base pairs.(9) Accordingly, in bacteria, a template transcribed in the presence of bacterial topoisomerase I, which only relaxes negative supercoils, becomes positively supercoiled,(…”

“Negative and positive supercoils are generated behind and ahead of advancing RNA polymerase, respectively (27).”

“5 and 100 kb (see level D in Table 1) that exist within larger 1-Mb loops, known as topologically associated domains (TADS) (level E in Table 1) (Gilbert and Allan 2014).”

“This result is likely relevant in vivo for eukaryotes [30, 36], since it implies that transcription itself can de-stabilise downstream nucleosomes, thereby facilitating further RNAP progress.”

“Further, DNA supercoiling is one of the most fundamental regulators of global gene expression in bacteria [122, 121], and was often shown to affect bacterial growth and transcription regulation [36, 132, 131].”

“,(35) is reinforced by accumulating evidence that highlights the role of the topoisomerase complex as a regulator of TAD boundaries.(42) Recent results demonstrate that topoisomerase II beta (TOP2B) binding overlaps with almost half of CTCF/cohesin binding sites, and that TOP2B may facilitate supercoiling at CTCF site…”

“) The global DNA supercoiling effects of these enzymes are in addition to the primarily local effects of gene transcription and DNA replication [12] and nucleoid structuring proteins [13].”

“In addition, DNA-binding proteins such as histones in eukaryotes and histone-like nucleoid proteins in bacteria sequester and maintain superhelical domains [33].”

“Furthermore, the structural configuration of chromatin in DNA forms a double-coil shape [26].”

“structures and stall RNA polymerases [222].”

“The first step of DNA compaction is its negative supercoiling (1–3), while further organization of the chromosome, referred to as a nucleoid, is aided by its association with nucleoid-associated proteins (NAPs) (4) and condensins (SMC proteins) (5).”

“…(in prokaryotes), topology and chromatin-processing DNA motors (level C in Table 1) and structural protein complexes which anchor the nuclear material to a fixed location within the cell (levels D and E in Table 1) are central to the structural organisation of all genomes (Gilbert and Allan 2014).”

“Plasmid replication is initiated in these assays from DnaC810 loading DnaB onto SSB-coated ssDNA generated at nicks or when DNA is supercoiled [33,34].”

“Supercoiling arises from the double helical properties of the DNA and the activities of DNA-associated machinery manipulating it 81,83 .”

“Possibly, this points to additional mechanisms of transcriptionally dependent chromatin folding not included in e-HiP-HoP, such as DNA supercoiling 25 or interactions with other nuclear structures (e.g., nuclear bodies 26 or the nuclear mesh 27 ).”

“The process of transcription will positively supercoil DNA in front of the polymerase and negatively supercoil it behind [5,71].”

“Single and double strand DNA breaks may also lead to a local release of topological constraints, a key component of the chromatin packing state [89,90].”

“This then undergoes supercoiling during cell division to form the metaphase chromosomes seen during mitosis and meiosis [4].”

“The contribution of DNA gyrase in maintaining the chromosome in an underwound state in bacterial cells can profoundly impact the binding of regulatory proteins, promoter firing dynamics, DNA replication, and chromosome architecture (4,19,20).”

“It has been suggested that nucleosome assembly and disassembly processes, through their modification of the local degree of supercoiling, can play important roles in gene regulation on distances exceeding several kb [38–42].”

“In cells, DNA unwinding is regulated by topoi-somerases 17 and has been linked to several physiological processes, such as gene expression 18,19 , chromatin structure 20,21 and DNA replication 22,23 and repair 24,25 .”

“Virtually any process that manipulates DNA strands can generate positive or negative DNA torsional stress (Esposito and Sinden, 1988; Baranello et al., 2012; Gilbert and Allan, 2014; Corless and Gilbert, 2016).”

“However, since chromatin is organized into 7 discrete domains that are topologically independent, the diffusion of supercoils may be 8 blocked and elevated levels of DNA supercoiling may build up (Gilbert and Allan, 2014).”

“tplecf 2lt lp fexp C ffiffi f p l+p lp ; (10)”

“DNA supercoiling is a tertiary nucleic acid structure, meaning that the molecular architecture of DNA exists in a self-twisted format.(1,2) The global alteration of supercoiled DNA structures is thought to be directly associated with its double helical properties.”

“DNA supercoiling is the over- or under-winding of the DNA double helix, which occurs as a consequence of polymerase activity and is modulated by topoisomerase activity [5].”

“The E. coli chromosome is organized by NAPs and additionally compac­ ted by unconstrained supercoiling (85, 106, 107).”

“coli cells with novobiocin inhibits the DNA gyrase activity and leads, as expected, to a reduction in the negative supercoiling level of plasmids [31] (Figure 2A–C).”

“…& Oda, 2010; Rampakakis, Gkogkas, Di Paola, & Zannis-Hadjopoulos, 2010; Roca, 2011), DNA gyrase (Corless & Gilbert, 2016; Rampakakis et al., 2010; Wang, 1996, 2002), chromatin-remodeling factors (Cairns, 2007; Corless & Gilbert, 2016), and histone acetyltransferase enzymes (Gilbert & Allan, 2014).”

“(Gilbert and Allan 2014) 2014 G4 motifs affect origin positioning and efficiency in two vertebrate replicators.”

“If loops are preformed into left-handed (or right-handed) inter-wound supercoils [52], pyramids are more sharply defined (Fig.”

“This discrepancy suggests that locus-specific factors, such as local chromatin accessibility or DNA secondary structure, may restrict recombinase access or compromise conjugate stability, thereby diminishing the benefits typically associated with short landing pads [ 56 , 57 ].”

“Assembly of nucleosomes is known to absorb negative supercoils (Gilbert & Allan, 2014; Koster, Crut, Shuman, Bjornsti, & Dekker, 2010; Patterton & von Holt, 1993), as well as stabilize the replication forks and thus securing the genomic stability (Prado & Maya, 2017).”

“These physical forces can arise from the activity of powerful revolving machines such as polymerases, the catalytic activity of chromatin remodelers, or the binding and bending activity of DNA-binding proteins [ 29 , 30 ].”

“Different mechanisms, like DNA supercoiling, aid in this process (Gilbert and Allan 2014), yet the packaging in certain viruses (Bryson et al. 2022; Irwin and Richards 2024), bacteria (Hocher et al. 2023), archaea (Pereira et al. 1997), and especially in eukaryotes (Khorasanizadeh 2004; Abouelmagd…”

“Approximately one (-) supercoil develops for each nucleosome removed.[10]”