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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We describe a new form of DNA repair heterogeneity along the genome. The repair rate of UV‐induced cyclobutane pyrimidine dimers (CPDs) was measured at single nucleotide resolution along the promoter and transcribed sequences of the human JUN gene in UV‐irradiated diploid fibroblasts. The promoter of this gene contains an array of sequence‐specific transcription factors located between nucleotides −200 and −50 relative to the major transcription start site. These sequences are repaired slowly; at many sites >50% of the CPDs are left unrepaired after 24 h. However, repair rates are 10‐fold faster near the transcription initiation site. This very fast repair is seen on both DNA strands between nucleotides −40 and +100 where at most positions >90% of the dimers are repaired within 4 h. There is a general gradient of repair efficiency of the transcribed DNA strand with faster repair within the 5′‐end and diminished repair towards the 3′‐end of the gene. The fast repair rates seen near the transcription initiation site may be explained by increased local concentrations of DNA repair factors that are associated with general transcription factors (e.g. TFIIH) functioning in transcription initiation. This domain‐specific DNA repair may aid in maintaining transcription initiation of essential genes after DNA damage.

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We have characterized the properties of immunopurified transcription complexes arrested at a specifically located cyclobutane pyrimidine dimer (CPD) using enzymatic probes and an in vitro transcription system with purified RNA polymerase II (RNAP II) and initiation factors. To help understand how RNAP II distinguishes between a natural impediment and a lesion in the DNA to initiate a repair event, we have compared the conformation of RNAP II complexes arrested at a CPD with complexes arrested at a naturally occurring elongation impediment. The footprint of RNAP II arrested at a CPD, using exonuclease III and T4 DNA polymerase's 335 exonuclease, covers ϳ35 base pairs and is asymmetrically located around the dimer. A similar footprint is observed when RNAP II is arrested at the human histone H3.3 arrest site. Addition of elongation factor SII to RNAP II arrested at a CPD produced shortened transcripts of discrete lengths up to 25 nucleotides shorter than those seen without SII. After addition of photolyase and exposure to visible light, some of the transcripts could be reelongated beyond the dimer, suggesting that SII-mediated transcript cleavage accompanied significant RNAP II backup, thereby providing access of the repair enzyme to the arresting CPD. Transcription-coupled repair (TCR)1 is a subpathway of nucleotide excision repair that removes lesions from the transcribed strand of actively transcribed genes (1). TCR has been shown to occur in mammalian cells (2), in Escherichia coli (3), and in Saccharomyces cerevisiae (4 -6). Several lines of evidence have suggested that an active RNA polymerase elongation complex is necessary for preferential repair of the transcribed strand (7). A model for TCR has been proposed in which the RNA polymerase stalled at a lesion directs repair enzymes to the transcribed strand of an active gene (2). This model assumes that the polymerase must be removed from the site of the lesion to provide access for the repair complex to the lesion site and to allow reannealing of the DNA strands to form a proper substrate for repair.An essential question about the mechanism of TCR is how the repair proteins recognize an RNAP II elongation complex arrested at a lesion and distinguish it from a transcription complex arrested at a natural arrest site. The process of transcriptional arrest can provide some clues to this question. RNAP II arrest sites have been identified and characterized (8).Arrest can occur at a bend in the helix axis of template DNA (9). It can also be induced by nucleotide depletion (10), DNA-binding drugs (11), and sequence-specific DNA-binding proteins (12). RNAP II can bypass arrest sites by activation of a cryptic endonuclease function that resides in the polymerase, a process mediated by the transcription elongation factor SII. SII-mediated transcript cleavage removes short oligonucleotides of discrete lengths from the 3Ј end of the nascent RNA. Transcript shortening is thought to restore the association of the 3Ј end of the transcript with the catalytic site in the po...