Torque-Induced Rotational Dynamics in Polymers: Torsional Blobs and Thinning

By using the blob theory and computer simulations, we investigate the properties of a linear polymer performing a stationary rotational motion around a long impenetrable rod. In particular, in the simulations the rotation is induced by a torque applied to the end of the polymer that is tethered to the rod. Three different regimes are found, in close analogy with the case of polymers pulled by a constant force at one end. For low torques the polymer rotates maintaining its equilibrium conformation. At intermediate torques the polymer assumes a trumpet shape, being composed by blobs of increasing size. At even larger torques the polymer is partially wrapped around the rod. We derive several scaling relations between various quantities as angular velocity, elongation and torque. The analytical predictions match the simulation data well. Interestingly, we find a "thinning" regime where the torque has a very weak (logarithmic) dependence on the angular velocity. We discuss the origin of this behavior, which has no counterpart in polymers pulled by an applied force.Comment: 30 pages, 8 figures, 1 TOC figure; video abstract at https://youtu.be/LwicoSkh3m

Inhibitory effect of a short Z-DNA forming sequence on transcription elongation by T7 RNA polymerase

DNA sequences capable of forming unusual secondary structures can be a source of genomic instability. In some cases that instability might be affected by transcription, as recently shown for the Z-DNA forming sequence (CG)14, which causes genomic instability both in mammalian cells and in bacteria, and this effect increases with its transcription. We have investigated the effect of this (CG)14 sequence on transcription with T7 RNA polymerase in vitro. We detected partial transcription blockage within the sequence; the blockage increased with negative supercoiling of the template DNA. This effect was not observed in a control self-complementary sequence of identical length and base composition as the (CG)14 sequence, when the purine–pyrimidine alternation required for Z-DNA formation was disrupted. These findings suggest that the inhibitory effect on T7 transcription results from Z-DNA formation in the (CG)14 sequence rather than from an effect of the sequence composition or from hairpin formation in either the DNA or the RNA product

DNA slip-outs cause RNA polymerase II arrest in vitro: potential implications for genetic instability

The abnormal number of repeats found in triplet repeat diseases arises from ‘repeat instability’, in which the repetitive section of DNA is subject to a change in copy number. Recent studies implicate transcription in a mechanism for repeat instability proposed to involve RNA polymerase II (RNAPII) arrest caused by a CTG slip-out, triggering transcription-coupled repair (TCR), futile cycles of which may lead to repeat expansion or contraction. In the present study, we use defined DNA constructs to directly test whether the structures formed by CAG and CTG repeat slip-outs can cause transcription arrest in vitro. We found that a slip-out of (CAG)20 or (CTG)20 repeats on either strand causes RNAPII arrest in HeLa cell nuclear extracts. Perfect hairpins and loops on either strand also cause RNAPII arrest. These findings are consistent with a transcription-induced repeat instability model in which transcription arrest in mammalian cells may initiate a ‘gratuitous’ TCR event leading to a change in repeat copy number. An understanding of the underlying mechanism of repeat instability could lead to intervention to slow down expansion and delay the onset of many neurodegenerative diseases in which triplet repeat expansion is implicated

Transcription Blockage by Bulky End Termini at Single-Strand Breaks in the DNA Template: Differential Effects of 5′ and 3′ Adducts

RNA polymerases from phage-infected bacteria and mammalian cells have been shown to bypass single-strand breaks (SSBs) with a single-nucleotide gap in the template DNA strand during transcription elongation; however, the SSB bypass efficiency varies significantly depending upon the backbone end chemistries at the break. Using a reconstituted T7 phage transcription system (T7 RNAP) and RNA polymerase II (RNAPII) in HeLa cell nuclear extracts, we observe a slight reduction in the level of transcription arrest at SSBs with no gap as compared to those with a single-nucleotide gap. We have shown that biotin and carbon-chain moieties linked to the 3′ side, and in select cases the 5′ side, of an SSB in the template strand strongly increase the level of transcription arrest when compared to unmodified SSBs. We also find that a small carbon-chain moiety linked to the upstream side of an SSB aids transcriptional bypass of SSBs for both T7 RNAP and RNAP II. Analysis of transcription across SSBs flanked by bulky 3′ adducts reveals the ability of 3′ end chemistries to arrest T7 RNAP in a size-dependent manner. T7 RNAP is also completely arrested when 3′ adducts or 3′-phosphate groups are placed opposite 5′-phosphate groups at an SSB. We have also observed that a biotinylated thymine in the template strand (without a break) does not pose a strong block to transcription. Taken together, these results emphasize the importance of the size of 3′, but usually not 5′, end chemistries in arresting transcription at SSBs, substantiating the notion that bulky 3′ lesions (e.g., topoisomerase cleavable complexes, 3′-phosphoglycolates, and 3′-unsaturated aldehydes) pose very strong blocks to transcribing RNA polymerases. These findings have implications for the processing of DNA damage through SSB intermediates and the mechanism of SSB bypass by T7 RNAP and mammalian RNAPII

Transcription Blockage by Bulky End Termini at Single-Strand Breaks in the DNA Template: Differential Effects of 5′ and 3′ Adducts

RNA polymerases from phage-infected bacteria and mammalian cells have been shown to bypass single-strand breaks (SSBs) with a single-nucleotide gap in the template DNA strand during transcription elongation; however, the SSB bypass efficiency varies significantly depending upon the backbone end chemistries at the break. Using a reconstituted T7 phage transcription system (T7 RNAP) and RNA polymerase II (RNAPII) in HeLa cell nuclear extracts, we observe a slight reduction in the level of transcription arrest at SSBs with no gap as compared to those with a single-nucleotide gap. We have shown that biotin and carbon-chain moieties linked to the 3′ side, and in select cases the 5′ side, of an SSB in the template strand strongly increase the level of transcription arrest when compared to unmodified SSBs. We also find that a small carbon-chain moiety linked to the upstream side of an SSB aids transcriptional bypass of SSBs for both T7 RNAP and RNAP II. Analysis of transcription across SSBs flanked by bulky 3′ adducts reveals the ability of 3′ end chemistries to arrest T7 RNAP in a size-dependent manner. T7 RNAP is also completely arrested when 3′ adducts or 3′-phosphate groups are placed opposite 5′-phosphate groups at an SSB. We have also observed that a biotinylated thymine in the template strand (without a break) does not pose a strong block to transcription. Taken together, these results emphasize the importance of the size of 3′, but usually not 5′, end chemistries in arresting transcription at SSBs, substantiating the notion that bulky 3′ lesions (e.g., topoisomerase cleavable complexes, 3′-phosphoglycolates, and 3′-unsaturated aldehydes) pose very strong blocks to transcribing RNA polymerases. These findings have implications for the processing of DNA damage through SSB intermediates and the mechanism of SSB bypass by T7 RNAP and mammalian RNAPII