Twist/Writhe Partitioning in a Coarse-Grained DNA Minicircle Model

Here we present a systematic study of supercoil formation in DNA minicircles under varying linking number by using molecular dynamics simulations of a two-bead coarse-grained model. Our model is designed with the purpose of simulating long chains without sacrificing the characteristic structural properties of the DNA molecule, such as its helicity, backbone directionality and the presence of major and minor grooves. The model parameters are extracted directly from full-atomistic simulations of DNA oligomers via Boltzmann inversion, therefore our results can be interpreted as an extrapolation of those simulations to presently inaccessible chain lengths and simulation times. Using this model, we measure the twist/writhe partitioning in DNA minicircles, in particular its dependence on the chain length and excess linking number. We observe an asymmetric supercoiling transition consistent with experiments. Our results suggest that the fraction of the linking number absorbed as twist and writhe is nontrivially dependent on chain length and excess linking number. Beyond the supercoiling transition, chains of the order of one persistence length carry equal amounts of twist and writhe. For longer chains, an increasing fraction of the linking number is absorbed by the writhe.Comment: 21 pages, 7 figures, 1 tabl

Divergent RNA transcription:A role in promoter unwinding?

New approaches using biotinylated-psoralen as a probe for investigating DNA structure have revealed new insights into the relationship between DNA supercoiling, transcription and chromatin compaction. We explore a hypothesis that divergent RNA transcription generates negative supercoiling at promoters facilitating initiation complex formation and subsequent promoter clearance

Predicting Knot or Catenane Type of Site-Specific Recombination Products

Site-specific recombination on supercoiled circular DNA yields a variety of knotted or catenated products. We develop a model of this process, and give extensive experimental evidence that the assumptions of our model are reasonable. We then characterize all possible knot or catenane products that arise from the most common substrates. We apply our model to tightly prescribe the knot or catenane type of previously uncharacterized data.Comment: 17 pages, 4 figures. Revised to include link to the companion paper, arXiv:0707.3896v1, that provides topological proofs underpinning the conclusions of the current paper. References update

Topological and Chromatin Alterations Influencing Genome Integrity

DNA topoisomerases Top1 and Top2 have redundant functions in resolving topological alterations arising during replication and transcription processes. Topoisomerases assist replication forks encountering transcription units, preventing chromosome fragility by minimizing the aberrant topological events. We investigated the role of topoisomerases in supercoil accumulation across the yeast genome using biotin tagged psoralen immunoprecipitation. We found that DNA is under-wound at gene boundaries and over-wound at transcribed regions. Top1 is associated with positively supercoiled chromatin as it accompanies RNA Polymerase II (Pol2) and its chromatin association is influenced by transcription levels of the individual genes. Top2 is associated with stable negative supercoiled chromatin at the gene boundaries, and its association is not dependent on transcription. Top2 promotes transcription efficiencies by forming gene loop structure and restricts Top1 and Pol2 leakage at gene boundaries. Ablation of Top2 protein decreases the negative supercoil accumulation at gene boundaries. Expression of E.coli TopA in topoisomerases double mutant in yeast (top2-1top1Δ) significantly resolves only the negative supercoil of gene boundaries and increases the accumulation of positive supercoil. The supercoil state at gene boundaries and ORFs are crucial for nucleosome occupancy. Using Hi-C techniques, we show that, centromeres are prominently interacting with other centromeres and the inter-chromosomal centromere interactions are depleted along with cohesin protein in top2-1top1Δ mutant expressing E.coli TopA. This work therefore summarizes the role of supercoil structures in preserving higher order architecture including nucleosome formation and chromosome organization

Controlled rotation mechanism of DNA strand exchange by the Hin serine recombinase.

DNA strand exchange by serine recombinases has been proposed to occur by a large-scale rotation of halves of the recombinase tetramer. Here we provide the first direct physical evidence for the subunit rotation mechanism for the Hin serine invertase. Single-DNA looping assays using an activated mutant (Hin-H107Y) reveal specific synapses between two hix sites. Two-DNA "braiding" experiments, where separate DNA molecules carrying a single hix are interwound, show that Hin-H107Y cleaves both hix sites and mediates multi-step rotational relaxation of the interwinding. The variable numbers of rotations in the DNA braid experiments are in accord with data from bulk experiments that follow DNA topological changes accompanying recombination by the hyperactive enzyme. The relatively slow Hin rotation rates, combined with pauses, indicate considerable rotary friction between synapsed subunit pairs. A rotational pausing mechanism intrinsic to serine recombinases is likely to be crucial for DNA ligation and for preventing deleterious DNA rearrangements

Gene clusters reflecting macrodomain structure respond to nucleoid perturbations

Focusing on the DNA-bridging nucleoid proteins Fis and H-NS, and integrating several independent experimental and bioinformatic data sources, we investigate the links between chromosomal spatial organization and global transcriptional regulation. By means of a novel multi-scale spatial aggregation analysis, we uncover the existence of contiguous clusters of nucleoid-perturbation sensitive genes along the genome, whose expression is affected by a combination of topological DNA state and nucleoid-shaping protein occupancy. The clusters correlate well with the macrodomain structure of the genome. The most significant of them lay symmetrically at the edges of the ter macrodomain and involve all of the flagellar and chemotaxis machinery, in addition to key regulators of biofilm formation, suggesting that the regulation of the physical state of the chromosome by the nucleoid proteins plays an important role in coordinating the transcriptional response leading to the switch between a motile and a biofilm lifestyle.Comment: Article: first 24 pages, 3 figures Supplementary methods: 1 page, 1 figure Supplementary results: 14 pages, 11 figure

Single-molecule observations of topotecan-mediated TopIB activity at a unique DNA sequence

The rate of DNA supercoil removal by human topoisomerase IB (TopIB) is slowed down by the presence of the camptothecin class of antitumor drugs. By preventing religation, these drugs also prolong the lifetime of the covalent TopIB–DNA complex. Here, we use magnetic tweezers to measure the rate of supercoil removal by drug-bound TopIB at a single DNA sequence in real time. This is accomplished by covalently linking camptothecins to a triple helix-forming oligonucleotide that binds at one location on the DNA molecule monitored. Surprisingly, we find that the DNA dynamics with the TopIB–drug interaction restricted to a single DNA sequence are indistinguishable from the dynamics observed when the TopIB–drug interaction takes place at multiple sites. Specifically, the DNA sequence does not affect the instantaneous supercoil removal rate or the degree to which camptothecins increase the lifetime of the covalent complex. Our data suggest that sequence-dependent dynamics need not to be taken into account in efforts to develop novel camptothecins

Recognition of DNA Supercoil Geometry by Mycobacterium tuberculosis Gyrase

Mycobacterium tuberculosis encodes only a single type II topoisomerase, gyrase. As a result, this enzyme likely carries out the cellular functions normally performed by canonical gyrase and topoisomerase IV, both in front of and behind the replication fork. In addition, it is the sole target for quinolone antibacterials in this species. Because quinolone-induced DNA strand breaks generated on positively supercoiled DNA ahead of replication forks and transcription complexes are most likely to result in permanent genomic damage, the actions of M. tuberculosis gyrase on positively supercoiled DNA were investigated. Results indicate that the enzyme acts rapidly on overwound DNA and removes positive supercoils much faster than it introduces negative supercoils into relaxed DNA. Canonical gyrase and topoisomerase IV distinguish supercoil handedness differently during the DNA cleavage reaction: while gyrase maintains lower levels of cleavage complexes on overwound DNA, topoisomerase IV maintains similar levels of cleavage complexes on both over- and underwound substrates. M. tuberculosis gyrase maintained lower levels of cleavage complexes on positively supercoiled DNA in the absence and presence of quinolone-based drugs. By retaining this important feature of canonical gyrase, the dual function M. tuberculosis type II enzyme remains a safe enzyme to act in front of replication forks and transcription complexes. Finally, the N-terminal gate region of the enzyme appears to be necessary to distinguish supercoil handedness during DNA cleavage, suggesting that the capture of the transport segment may influence how gyrase maintains cleavage complexes on substrates with different topological states