Topology and kinetics of R-loop formation.

R-loops are structures containing an RNA-DNA duplex and an unpaired DNA strand. They can be formed upon "invasion" of an RNA strand into a DNA duplex, during which the RNA displaces the homologous DNA strand and binds the complementary strand. R-loops have many significant beneficial or deleterious biological effects, so it is important to understand the mechanisms for their generation and processing. We propose a model for co-transcriptional R-loop formation, in which their generation requires passage of the nascent RNA "tail" through the gap between the separated DNA strands. This passage becomes increasingly difficult with lengthening of the RNA tail. The length of the tail increases upon increasing distance between the transcription start site and the site of R-loop initiation. This causes reduced yields of R-loops with greater distance from the transcription start site. However, alternative pathways for R-loop formation are possible, involving either transient disruption of the transcription complex or the hypothetical formation of a triple-stranded structure, as a "collapsed R-loop." These alternative pathways could account for the fact that in many systems R-loops are observed very far from the transcription start site. Our model is consistent with experimental data and makes general predictions about the kinetics of R-loop formation.

Effects of isolated nonspecific binders upon the search for specific targets: Absolute rates versus competition between the targets.

Many biological processes involve macromolecules searching for their specific targets that are surrounded by other objects, and binding to these objects affects the target search. Acceleration of the target search by nonspecific binders was observed experimentally and analyzed theoretically, for example, for DNA-binding proteins. According to existing theories this acceleration requires continuous transfer between the nonspecific binders and the specific target. In contrast, our analysis predicts that (i) nonspecific binders could accelerate the search without continuous transfer to the specific target provided that the searching particle is capable of sliding along the binder; (ii) in some cases such binders could decelerate the target search, but provide an advantage in competition with the "binder-free" target; (iii) nonbinding objects decelerate the target search. We also show that although the target search in the presence of binders could be considered as diffusion in inhomogeneous media, in the general case it cannot be described by the effective diffusion coefficient.

Transcription Inhibition by PNA-Induced R-Loops.

R-loops are structures consisting of an RNA-DNA duplex and an unpaired DNA strand. They can form during transcription upon nascent RNA "threadback" invasion into the DNA duplex to displace the non-template DNA strand. R-loops occur naturally in all kingdoms of life, and they have multiple biological effects. Therefore, it is of interest to study the artificial induction of R-loops and to monitor their effects in model in vitro systems to learn mechanisms. Here we describe transcription blockage in vitro by R-loop formation induced by peptide nucleic acid (PNA) binding to the non-template DNA strand.

R-loop generation during transcription: Formation, processing and cellular outcomes.

R-loops are structures consisting of an RNA-DNA duplex and an unpaired DNA strand. They can form during transcription upon nascent RNA "threadback" invasion into the DNA duplex to displace the non-template strand. Although R-loops occur naturally in all kingdoms of life and serve regulatory roles, they are often deleterious and can cause genomic instability. Of particular importance are the disastrous consequences when replication forks or transcription complexes collide with R-loops. The appropriate processing of R-loops is essential to avoid a number of human neurodegenerative and other clinical disorders. We provide a perspective on mechanistic aspects of R-loop formation and their resolution learned from studies in model systems. This should contribute to improved understanding of R-loop biological functions and enable their practical applications. We propose the novel employment of artificially-generated stable R-loops to selectively inactivate tumor cells.

A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system.

The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations. One of these possibilities arises when an additional transcription initiation site (e.g. secondary promoter) is present upstream from the primary promoter of the target gene. In this case, transcription inhibition might be achieved by inducing the formation of an RNA-DNA hybrid (R-loop) upon transcription from the secondary promoter. The R-loop could extend into the region of the primary promoter, to interfere with promoter recognition by RNA polymerase and thereby inhibit transcription. As a sequence-specific R-loop-inducing agent, a peptide nucleic acid (PNA) could be designed to facilitate R-loop formation by sequestering the non-template DNA strand. To investigate this mode for transcription inhibition, we have employed a model system in which a PNA binding site is localized between the T3 and T7 phage RNA polymerase promoters, which respectively assume the roles of primary and secondary promoters. In accord with our model, we have demonstrated that with PNA-bound DNA substrates, transcription from the T7 promoter reduces transcription from the T3 promoter by 30-fold, while in the absence of PNA binding there is no significant effect of T7 transcription upon T3 transcription.

Strong transcription blockage mediated by R-loop formation within a G-rich homopurine-homopyrimidine sequence localized in the vicinity of the promoter.

Guanine-rich (G-rich) homopurine-homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence. Remarkably, the blockage is not pronounced if transcription is performed in the presence of RNase H, which specifically digests the RNA strands within RNA-DNA hybrids. The blockage also becomes less pronounced upon reduced RNA polymerase concentration. Based upon these observations and those from control experiments, we conclude that the blockage is primarily due to the formation of stable RNA-DNA hybrids (R-loops), which inhibit successive rounds of transcription. Our results could be relevant to transcription dynamics in vivo (e.g. transcription 'bursting') and may also have practical implications for the design of expression vectors.

When transcription goes on Holliday: Double Holliday junctions block RNA polymerase II transcription in vitro.

Non-canonical DNA structures can obstruct transcription. This transcription blockage could have various biological consequences, including genomic instability and gratuitous transcription-coupled repair. Among potential structures causing transcription blockage are Holliday junctions (HJs), which can be generated as intermediates in homologous recombination or during processing of stalled replication forks. Of particular interest is the double Holliday junction (DHJ), which contains two HJs. Topological considerations impose the constraint that the total number of helical turns in the DNA duplexes between the junctions cannot be altered as long as the flanking DNA duplexes are intact. Thus, the DHJ structure should strongly resist transient unwinding during transcription; consequently, it is predicted to cause significantly stronger blockage than single HJ structures. The patterns of transcription blockage obtained for RNA polymerase II transcription in HeLa cell nuclear extracts were in accordance with this prediction. However, we did not detect transcription blockage with purified T7 phage RNA polymerase; we discuss a possible explanation for this difference. In general, our findings implicate naturally occurring Holliday junctions in transcription arrest.

Torque-winding interdependence for a flexible polymer chain wound around a cylinder in the presence of obstacles.

Polymer chains winding around each other or around other objects occur in many natural systems; the physical consequences of this winding are therefore of significant interest. A polymer chain could be surrounded by various bulky objects (referred as obstacles), such as other macromolecules or macromolecular aggregates. Here we show that for a long flexible polymer chain wound around a cylinder, the presence of obstacles could modify the winding-torque interdependence, in some cases leading to phase-transition-like behavior in which the winding occurs only when the torque exceeds some critical value. Possible implications of this effect are discussed in relation to the biophysics of nucleic acids.

Transcription blockage by stable H-DNA analogs in vitro.

DNA sequences that can form unusual secondary structures are implicated in regulating gene expression and causing genomic instability. H-palindromes are an important class of such DNA sequences that can form an intramolecular triplex structure, H-DNA. Within an H-palindrome, the H-DNA and canonical B-DNA are in a dynamic equilibrium that shifts toward H-DNA with increased negative supercoiling. The interplay between H- and B-DNA and the fact that the process of transcription affects supercoiling makes it difficult to elucidate the effects of H-DNA upon transcription. We constructed a stable structural analog of H-DNA that cannot flip into B-DNA, and studied the effects of this structure on transcription by T7 RNA polymerase in vitro. We found multiple transcription blockage sites adjacent to and within sequences engaged in this triplex structure. Triplex-mediated transcription blockage varied significantly with changes in ambient conditions: it was exacerbated in the presence of Mn(2+) or by increased concentrations of K(+) and Li(+). Analysis of the detailed pattern of the blockage suggests that RNA polymerase is sterically hindered by H-DNA and has difficulties in unwinding triplex DNA. The implications of these findings for the biological roles of triple-stranded DNA structures are discussed.

PNA binding to the non-template DNA strand interferes with transcription, suggesting a blockage mechanism mediated by R-loop formation.

Peptide Nucleic Acids (PNAs) are artificial DNA mimics with superior nucleic acid binding capabilities. T7 RNA polymerase (T7 RNAP) transcription upon encountering PNA bound to the non-template DNA strand was studied in vitro. A characteristic pattern of blockage signals was observed, extending downstream from the PNA binding site, similar to that produced by G-rich homopurine-homopyrimidine (hPu-hPy) sequences and likely caused by R-loop formation. Since blocked transcription complexes in association with stable R-loops may interfere with replication and in some cases trigger apoptosis, targeted R-loop formation might be employed to inactivate selected cells, such as those in tumors, based upon their unique complement of expressed genes.

Relationships between the winding angle, the characteristic radius, and the torque for a long polymer chain wound around a cylinder: implications for RNA winding around DNA during transcription.

Long polymer chains are ubiquitous in biological systems and their mechanical properties have significant impact upon biological processes. Of particular interest is the situation in which polymer chains are wound around each other or around other objects. We have analyzed the parameters of a long Gaussian polymer chain wound around a cylinder as a function of the torque applied to the ends of the chain. We have shown that for sufficiently long polymer chains, an average winding angle and a characteristic radius of the chain can be determined from a modified Bessel function of purely imaginary order, in which the value of the order is equivalent to the applied torque, normalized to the product of the absolute temperature and the Boltzmann constant. The obtained results are consistent with a simplified interpretation in terms of "torsional blobs," and this could be extended to nonideal chains with excluded volumes. We have also extended our results to the case of a polymer chain rotating in viscous medium. Our results could be used to estimate the mechanical strains that appear in DNA and RNA during transcription, as these might initiate formation of unusual DNA structures, invasion of RNA into the DNA duplex (R-loop formation), and modulation of the interactions of DNA and RNA with proteins.

Transcription blockage by homopurine DNA sequences: role of sequence composition and single-strand breaks.

The ability of DNA to adopt non-canonical structures can affect transcription and has broad implications for genome functioning. We have recently reported that guanine-rich (G-rich) homopurine-homopyrimidine sequences cause significant blockage of transcription in vitro in a strictly orientation-dependent manner: when the G-rich strand serves as the non-template strand [Belotserkovskii et al. (2010) Mechanisms and implications of transcription blockage by guanine-rich DNA sequences., Proc. Natl Acad. Sci. USA, 107, 12816-12821]. We have now systematically studied the effect of the sequence composition and single-stranded breaks on this blockage. Although substitution of guanine by any other base reduced the blockage, cytosine and thymine reduced the blockage more significantly than adenine substitutions, affirming the importance of both G-richness and the homopurine-homopyrimidine character of the sequence for this effect. A single-strand break in the non-template strand adjacent to the G-rich stretch dramatically increased the blockage. Breaks in the non-template strand result in much weaker blockage signals extending downstream from the break even in the absence of the G-rich stretch. Our combined data support the notion that transcription blockage at homopurine-homopyrimidine sequences is caused by R-loop formation.

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.

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.

Anchoring nascent RNA to the DNA template could interfere with transcription.

During normal transcription, the nascent RNA product is released from the DNA template. However, in some cases, the RNA remains bound or can become reattached to the template DNA duplex (for example, through R-loop formation). We have analyzed the effect on transcription elongation of nascent RNA anchoring to the template DNA duplex. Because the RNA polymerase follows a helical path along DNA duplex during transcription, the anchoring would result in wrapping the nascent RNA around the DNA in the region between the anchoring point and the translocating polymerase. This wrapping would cause an unfavorable loss of conformation entropy of the nascent RNA. It consequently would create an apparent force to unwrap the RNA by disrupting either the transcription complex or the anchoring structure. We have estimated that this force would be comparable to those required to melt nucleic acid duplexes or to arrest transcription elongation in single-molecule experiments. We predict that this force would create negative supercoiling in the DNA duplex region between the anchoring point and the transcribing RNA polymerase: this can promote the formation of unusual DNA structures and facilitate RNA invasion into the DNA duplex. Potential biological consequences of these effects are discussed.

Mechanisms and implications of transcription blockage by guanine-rich DNA sequences.

Various DNA sequences that interfere with transcription due to their unusual structural properties have been implicated in the regulation of gene expression and with genomic instability. An important example is sequences containing G-rich homopurine-homopyrimidine stretches, for which unusual transcriptional behavior is implicated in regulation of immunogenesis and in other processes such as genomic translocations and telomere function. To elucidate the mechanism of the effect of these sequences on transcription we have studied T7 RNA polymerase transcription of G-rich sequences in vitro. We have shown that these sequences produce significant transcription blockage in an orientation-, length- and supercoiling-dependent manner. Based upon the effects of various sequence modifications, solution conditions, and ribonucleotide substitutions, we conclude that transcription blockage is due to formation of unusually stable RNA/DNA hybrids, which could be further exacerbated by triplex formation. These structures are likely responsible for transcription-dependent replication blockage by G-rich sequences in vivo.

Peptide nucleic acid (PNA) binding and its effect on in vitro transcription in friedreich's ataxia triplet repeats.

Peptide nucleic acids (PNAs) are DNA mimics in which peptide-like linkages are substituted for the phosphodiester backbone. Homopyrimidine PNAs can invade double-stranded DNA containing the homologous sequence by displacing the homopyrimidine strand from the DNA duplex and forming a PNA/DNA/PNA triplex with the complementary homopurine strand. Among biologically interesting targets for triplex-forming PNA are (GAA/CTT)(n) repeats. Expansion of these repeats results in partial inhibition of transcription in the frataxin gene, causing Friedreich's ataxia. We have studied PNA binding and its effect on T7 RNA polymerase transcription in vitro for short repeats (n = 3) and for long repeats (n = 39), placed in both possible orientations relative to the T7 promoter such that either the GAA-strand, or the CTT-strand serves as the template for transcription. In all cases PNA bound specifically and efficiently to its target sequence. For the short insert, PNA binding to the template strand caused partial transcription blockage with well-defined sites of RNA product truncation in the region of the PNA-binding sequence, whereas binding to the nontemplate strand did not block transcription. However, PNA binding to long repeats, whether in the template or the nontemplate strand, resulted in a dramatic reduction of the amount of full-length transcription product, although in the case of the nontemplate strand there were no predominant truncation sites. Biological implications of these results are discussed.

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.