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 homopurinehomopyrimidine 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-, lengthand 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.R-loops | DNA supercoiling | Hoogsteen base pairing | inosine | 7-deazaquanosine S equence-specific modulation of transcription, including transcription blockage or impediment, plays an important role in DNA transactions, for example, transcription-related mutagenesis and recombination (reviewed in refs. 1 and 2) and could also be responsible for several severe genetic diseases (reviewed in refs. 3-5).Among the DNA sequences that could affect transcription are GC-rich homopurine-homopyrimidine (hPu/hPy) stretches. These sequences could form unusual DNA structures, including triplexes and G quadruplexes (reviewed in refs. 3-5), which have been implicated in several transcription-dependent phenomena (for example, see refs. 6-9).Another important property of these sequences is a dramatic asymmetry in the stabilities of RNA/DNA duplexes: The rPu/dPy duplex is significantly more stable, whereas the rPy/dPu duplex is less stable than a DNA/DNA duplex of the same sequence (10). The increased stability of rPu/dPy duplexes is likely responsible for stable R-loop formation by these sequences (11), although alternative DNA structures might also be involved (8,12,13).The simplest example of GC-rich hPu/hPy sequences, the G n ∕C n repeats, is abundant in various genomes, including transcribed domains (14, 15).The G 32 ∕C 32 stretch was previously shown to stall DNA replication in Escherichia coli plasmids in vivo (16). Remarkably, this effect was observed only when the sequence was transcribed, which led to a model stipulating that this sequence stalled an elongating RNA polymerase, and the stalled transcription complex, in turn, blocked the replication machinery (16).To elucidate the mechanism of transcription blockage by this sequence, we have studied its effect on T7 RNA polymerase (T7 RNAP) transcription in vitro, using various sequence modifications and solution conditions that allowed us to discriminate between possible D...

Ultraviolet light has been linked with the development of human skin cancers. Such cancers often exhibit mutations in the p53 tumor suppressor gene. Ligation-mediated polymerase chain reaction was used to analyze at nucleotide resolution the repair of cyclobutane pyrimidine dimers along the p53 gene in ultraviolet-irradiated human fibroblasts. Repair rates at individual nucleotides were highly variable and sequence-dependent. Slow repair was seen at seven of eight positions frequently mutated in skin cancer, suggesting that repair efficiency may strongly contribute to the mutation spectrum in a cancer-associated gene.

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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 nucleasesensitive element of the human c-MYC promoter, which is mutagenic in mammalian cells (Wang, G., and Vasquez, K. M. 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 transcriptioninduced triplex formation enhances pre-existing weak transcription pause sites within the flanking sequences by creating steric obstacles for the transcription machinery.In addition to its primary function in protein synthesis, transcription plays an important role in gene regulation and genome modification. Transcription through a particular DNA region can also increase the rate of mutation in this region (transcription-assisted mutagenesis) or create a hot spot for homologous recombination (transcription-assisted recombination) (reviewed in Ref. 1). Both of these phenomena can occur as a consequence of the DNA opening during transcription, because single-stranded DNA is more sensitive than double-stranded DNA to attack from a number of agents, including some DNA-modifying enzymes. In some cases, transcriptioninduced mutagenesis is enhanced by the formation of an unusually stable RNA-DNA hybrid and/or secondary structure in the nontemplate strand, both of which stabilize R-loops, thus prolonging DNA opening (for example see Refs. 2, 3). These two pathways, however, are not necessarily associated with transcription pausing or arrest.A special pathway of DNA processing associated with transcription arrest is transcription-coupled DNA repair (TCR) 3 (see Refs. 4, 5 and reviewed in Refs. 6 -9). TCR manifests itself as a preferential repair of DNA lesions (for example pyrimidine photodimers) in the template strand versus the nontemplate strand. This sub-pathway of nucleotide excision repair involves dedicated enzymatic machinery to displace the RNA polymerase and to provide an accelerated recovery of functionally important genes as well as clearing the DNA template for replication and other transactions. According to our current model for TCR, when RNA polymerase becomes arrested upon encountering a lesion in the template strand, the arrested RNA polymerase interacts with specific TCR factors, and this interaction serves as a signal for excision of the l...

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