Boris P. Belotserkovskii

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. We have studied T7 RNA polymerase transcription through the sequence from the nuclease-sensitive element of the human c-MYC promoter, which is mutagenic in mammalian cells ( Wang, G., and Vasquez, K. M. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 13448-13453 ). This element has two mirror-symmetric homopurine-homopyrimidine blocks that potentially can form either DNA triplex (H-DNA) or quadruplex structures. We detected truncated transcription products indicating partial transcription arrest within and closely downstream of the element. The arrest required negative supercoiling and was much more pronounced when the pyrimidine-rich strand of the element served as the template. The exact positions of arrest sites downstream from the element depended upon the downstream flanking sequences. We made various nucleotide substitutions in the wild-type sequence from the c-MYC nuclease-sensitive element that specifically destabilize either the triplex or the quadruplex structure. When these substitutions were ranked for their effects on transcription, the results implicated the triplex structure in the transcription arrest. We suggest that transcription-induced triplex formation enhances pre-existing weak transcription pause sites within the flanking sequences by creating steric obstacles for the transcription machinery.

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.

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 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.

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.

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.

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 Friedreichs 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.

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

Abstract 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.

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. We varied the length and the composition of the reacting DNA segments. The minimal DNA duplex length at which we detected catenation was 50–60 bp for DNA gyrase and 40 bp for topoisomerase IV (Topo IV). For Topo IV, catenation was observed when one, but not both, of the DNA-DNA duplexes was replaced by a DNA-RNA duplex. Topo IV cleaved the DNA-DNA duplex, but not the DNA-RNA duplex implying that the DNA-RNA duplex can be a T-segment but not a G-segment.