(CGG)n.(CCG)n and (CTG)n.(CAG)n repeats of varying length were cloned into a bacterial plasmid, and the progression of the replication fork through these repeats was followed using electrophoretic analysis of replication intermediates. We observed stalling of the replication fork within repeated DNAs and found that this effect depends on repeat length, repeat orientation relative to the replication origin and the status of protein synthesis in a cell. Interruptions within repeated DNAs, similar to those observed in human genes, abolished the replication blockage. Our results suggest that the formation of unusual DNA structures by trinucleotide repeats in the lagging-strand template may account for the observed replication blockage and have relevance to repeat expansion in humans.

The mechanisms of trinucleotide repeat expansions, underlying more than a dozen hereditary neurological disorders, are yet to be understood. Here we looked at the replication of (CGG) n ⅐ (CCG) n and (CAG) n ⅐ (CTG) n repeats and their propensity to expand in Saccharomyces cerevisiae. Using electrophoretic analysis of replication intermediates, we found that (CGG) n ⅐ (CCG) n repeats significantly attenuate replication fork progression. Replication inhibition for this sequence becomes evident at as few as ϳ10 repeats and reaches a maximal level at 30 to 40 repeats. This is the first direct demonstration of replication attenuation by a triplet repeat in a eukaryotic system in vivo. For (CAG) n ⅐ (CTG) n repeats, on the contrary, there is only a marginal replication inhibition even at 80 repeats. The propensity of trinucleotide repeats to expand was evaluated in a parallel genetic study. In wild-type cells, expansions of (CGG) 25 ⅐ (CCG) 25 and (CAG) 25 ⅐ (CTG) 25 repeat tracts occurred with similar low rates. A mutation in the large subunit of the replicative replication factor C complex (rfc1-1) increased the expansion rate for the (CGG) 25 repeat ϳ50-fold but had a much smaller effect on the expansion of the (CTG) 25 repeat. These data show dramatic sequence-specific expansion effects due to a mutation in the lagging strand DNA synthesis machinery. Together, the results of this study suggest that expansions are likely to result when the replication fork attempts to escape from the stall site.Trinucleotide repeats, specifically (CGG) n ⅐ (CCG) n , (CAG) n ⅐ (CTG) n , and (GAA) n ⅐ (TTC) n , have attracted wide attention since their expansion leads to numerous hereditary neurological disorders in humans, including fragile X syndrome, Huntington's disease, myotonic dystrophy, Friedreich's ataxia, etc. (reviewed in reference 49). The inheritance of these diseases is characterized by the so-called anticipation, i.e., an increase in the probability, onset, and the severity of a disease as it passes through generations. The molecular basis for anticipation is that trinucleotide repeats are stably inherited and cause no harm unless the number of repeats exceeds a threshold of roughly 25, upon which an intergenerational transmission of expanded versions of these repeats becomes progressively more common (reviewed in reference 1).The mechanisms responsible for trinucleotide repeats expansion remain unclear. The largest volume of data supports an idea that abnormal replication of repeated stretches is responsible for their expansion. First, it is generally believed that the length dependence of expansion is linked to the ability of repeated DNAs to form unusual secondary structures, since the stability of such structures increases with repeats' lengths (reviewed in reference 27). Formation of these unusual DNA structures by trinucleotide repeats significantly compromises DNA polymerization in vitro (11,21,47). This polymerization blockage facilitates occasional misalignment between the newly synthesized and the template D...

The influence of d(G) n ·d(C) n repeats on plasmid replication in Escherichia coli cells was analyzed using electrophoretic analysis of replication intermediates. These repeats impeded the replication fork in a lengthand orientation-dependent manner. Unexpectedly, the replication arrest relied primarily on the repeats' transcription. When the d(C) n sequence served as the transcriptional template, both transcription and replication were blocked. This was true for transcription driven by either bacterial or phage RNA polymerases. We hypothesize that the replication fork halts after it encounters a stalled ternary complex of the RNA polymerase, the DNA template and the r(G) n transcript. This constitutes a novel mechanism for the regulation of replication elongation. The effects of this mechanism on repeat length polymorphism and genome rearrangements are discussed.

Studying the activity of T7 DNA polymerase (Sequenase) on open circular DNAs, we observed virtually complete termination within potential triplex‐forming sequences. Mutations destroying the triplex potential of the sequences prevented termination, while compensatory mutations restoring triplex potential restored it. We hypothesize that strand displacement during DNA polymerization of double‐helical templates brings three DNA strands (duplex DNA downstream of the polymerase plus a displaced overhang) into close proximity, provoking triplex formation, which in turn prevents further DNA synthesis. Supporting this idea, we found that Sequenase is unable to propagate through short triple‐helical stretches within single‐stranded DNA templates. Thus, DNA polymerase, by inducing triplex formation at specific sequences in front of the replication fork, causes self‐termination. Possible biological implications of such ‘conformational suicide’ are discussed. Our data also provide a novel way to target DNA polymerases at specific sequences using triplex‐forming oligonucleotides.

Homopurine-homopyrimidine mirror repeats are known to form Intramolcular DNA triplexes in Wiro.By probing with chemical agents specific for unusual DNA conformations, we have now demonstrated the formation of intramolecular triplexes consiting of G G-C and TOAST base triplets by DNA sequences that are neither homopurinehomopyrimkdine nor mirror repeats. This finding s ntly enlarges the number of sequences that could form DNA triplexes. The observed triplexes are stable under the conditions that are optimal for DNA polymerases in vitro. We found that triplex formation causes specific termination of DNA polymerization in viro. This effect is detected for diferent DNA polymerases and may have Implications for the ruion of DNA replication in vivo.