The mechanism by which trinucleotide expansion occurs in human genes is not understood. However, it has been hypothesized that DNA secondary structure may actively participate by preventing FEN-1 cleavage of displaced Okazaki fragments. We show here that secondary structure can, indeed, play a role in expansion by a FEN-1-dependent mechanism. Secondary structure inhibits flap processing at CAG, CGG, or CTG repeats in a length-dependent manner by concealing the 5' end of the flap that is necessary for both binding and cleavage by FEN-1. Thus, secondary structure can defeat the protective function of FEN-1, leading to site-specific expansions. However, when FEN-1 is absent from the cell, alternative pathways to simple inhibition of flap processing contribute to expansion.

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...

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Trinucleotide repeats (TNRs) undergo high frequency mutagenesis to cause at least 15 neurodegenerative diseases. To understand better the molecular mechanisms of TNR instability in cultured cells, a new genetic assay was created using a shuttle vector. The shuttle vector contains a promoter-TNR-reporter gene construct whose expression is dependent on TNR length. The vector harbors the SV40 ori and large T antigen gene, allowing portability between primate cell lines. The shuttle vector is propagated in cultured cells, then recovered and analyzed in yeast using selection for reporter gene expression. We show that (CAG•CTG)25−33 contracts at frequencies as high as 1% in 293T and 293 human cells and in COS-1 monkey cells, provided that the plasmid undergoes replication. Hairpin-forming capacity of the repeat sequence stimulated contractions. Evidence for a threshold was observed between 25 and 33 repeats in COS-1 cells, where contraction frequencies increased sharply (up 720%) over a narrow range of repeat lengths. Expression of the mismatch repair protein Mlh1 does not correlate with repeat instability, suggesting contractions are independent of mismatch repair in our system. Together, these findings recapitulate certain features of human genetics and therefore establish a novel cell culture system to help provide new mechanistic insights into CAG•CTG repeat instability.

Vertebrates have developed multiple mechanisms to coordinate the replication of epigenetic and genetic information. Dnmt1 encodes the maintenance enzyme DNA-methyltransferase, which is responsible for propagating the DNA methylation pattern and the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5 region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequence elements associated with previously characterized origins of DNA replication. This study tests the hypothesis that the dnmt1 region containing these elements is an origin of replication in human cells. First, we demonstrate that a vector containing this dnmt1 sequence is able to support autonomous replication when transfected into HeLa cells. Second, using a gel retardation assay, we show that it contains a site for binding of origin-rich sequences binding activity, a recently purified replication protein. Finally, using competitive polymerase chain reaction, we show that replication initiates in this region in vivo. Based on these lines of evidence, we propose that initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.