A model for transcription-dependent R-loop formation at double-stranded DNA breaks: Implications for their detection and biological effects.

R-loops are structures containing an RNA-DNA duplex and an unpaired DNA strand. During R-loop formation an RNA strand invades the DNA duplex, displacing the homologous DNA strand and binding the complementary DNA strand. Here we analyze a model for transcription-dependent R-loop formation at double-stranded DNA breaks (DSBs).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A triplex-forming sequence from the human c-MYC promoter interferes with DNA transcription.

Naturally occurring DNA sequences that are able to form unusual DNA structures have been shown to be mutagenic, and in some cases the mutagenesis induced by these sequences is enhanced by their transcription. It is possible that transcription-coupled DNA repair induced at sites of transcription arrest might be involved in this mutagenesis. Thus, it is of interest to determine whether there are correlations between the mutagenic effects of such noncanonical DNA structures and their ability to arrest transcription.

Topoisomerase action on short DNA duplexes reveals requirements for gate and transfer DNA segments.

Type II topoisomerases change DNA topology by passage of one DNA duplex (the transfer, T-segment) through a transient double-stranded break in another (the gate, G-segment). Here we monitor the passage between short double-stranded DNA segments within long single-stranded DNA circles that leads to catenation of the circles. To facilitate catenation, the circles were brought into close proximity using a tethering oligonucleotide, which was removed after the reaction was complete.

The Saccharomyces cerevisiae Smc2/4 condensin compacts DNA into (+) chiral structures without net supercoiling.

Smc2/4 forms the core of the Saccharomyces cerevisiae condensin, which promotes metaphase chromosome compaction. To understand how condensin manipulates DNA, we used two in vitro assays to study the role of SMC (structural maintenance of chromosome) proteins and ATP in reconfiguring the path of DNA. The first assay evaluated the topology of knots formed in the presence of topoisomerase II.

Analysis of a one-dimensional random walk with irreversible losses at each step: applications for protein movement on DNA.

We analysed a one-dimensional random walk between two points when the migrating particle could be irreversibly lost (dissociated) from the system at each step of the process. We show that in the case of losses at each step the average number of steps made by the particle that reaches the final point does not obey quadratic dependence on the distance between the starting and the final points: for long distances this dependence is linear. This is because losses "select" for shorter pathways between the starting and the final points.