Mismatch repair stabilizes the cellular genome by correcting DNA replication errors and by blocking recombination events between divergent DNA sequences. The reaction responsible for strand-specific correction of mispaired bases has been highly conserved during evolution, and homologs of bacterial M u 6 and Mu& which play key roles in mismatch recognition and initiation of repair, have been identified in yeast and mammalian cells. Inactivation of genes encoding these activities results in a large increase in spontaneous mutability, and in the case of mice and men, predisposition to tumor development. CONTENTS

A quantitative and selective genetic assay was developed to monitor expansions of trinucleotide repeats (TNRs) in yeast. A promoter containing 25 repeats allows expression of a URA3 reporter gene and yields sensitivity to the drug 5-f luoroorotic acid. Expansion of the TNR to 30 or more repeats turns off URA3 and provides drug resistance. When integrated at either of two chromosomal loci, expansion rates were 1 ؋ 10 ؊5 to 4 ؋ 10 ؊5 per generation if CTG repeats were replicated on the lagging daughter strand. PCR analysis indicated that 5-28 additional repeats were present in 95% of the expanded alleles. No significant changes in CTG expansion rates occurred in strains deficient in the mismatch repair gene MSH2 or the recombination gene RAD52. The frequent nature of CTG expansions suggests that the threshold number for this repeat is below 25 in this system. In contrast, expansions of the complementary repeat CAG occurred at 500-to 1,000-fold lower rates, similar to a randomized (C,A,G) control sequence. When the reporter plasmid was inverted within the chromosome, switching the leading and lagging strands of replication, frequent expansions were observed only when CTG repeats resided on the lagging daughter strand. Among the rare CAG expansions, the largest gain in tract size was 38 repeats. The control repeats CTA and TAG showed no detectable rate of expansions. The orientationdependence and sequence-specificity data support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments.

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...

Recent studies have shown that Saccharomyces cerevisiae Msh2p and Msh6p form a complex that specifically binds to DNA containing base pair mismatches. In this study, we performed a genetic and biochemical analysis of the Msh2p-Msh6p complex by introducing point mutations in the ATP binding and putative helix-turn-helix domains of MSH2. The effects of these mutations were analyzed genetically by measuring mutation frequency and biochemically by measuring the stability, mismatch binding activity, and ATPase activity of msh2p (mutant msh2p)-Msh6p complexes. A mutation in the ATP binding domain of MSH2 did not affect the mismatch binding specificity of the msh2p-Msh6p complex; however, this mutation conferred a dominant negative phenotype when the mutant gene was overexpressed in a wild-type strain, and the mutant protein displayed biochem ical defects consistent with defects in mismatch repair downstream of mismatch recognition. Helix-turn-helix domain mutant proteins displayed two different properties. One class of mutant proteins was defective in forming complexes with Msh6p and also failed to recognize base pair mismatches. A second class of mutant proteins displayed properties similar to those observed for the ATP binding domain mutant protein. Taken together, these data suggested that the proposed helix-turn-helix domain of Msh2p was unlikely to be involved in mismatch recognition. We propose that the MSH2 helix-turn-helix domain mediates changes in Msh2p-Msh6p interactions that are induced by ATP hydrolysis; the net result of these changes is a modulation of mismatch recognition.Mismatch repair is a highly conserved process that results in the removal of base pair mismatches that occur during DNA replication, DNA damage, and genetic recombination (25,37,45). Much of our understanding of mismatch repair comes from Escherichia coli, where an in vitro repair reaction was developed and individual components were identified and purified to homogeneity (28,36). This approach led to the identification of three proteins, MutS, MutL, and MutH, that were shown to be important for the initial steps in mismatch repair. These steps are thought to involve MutS binding to the base pair mismatch, followed by ATP-dependent binding of MutL to the MutS-mismatch complex (15). This complex is then thought to activate MutH, an endonuclease that cleaves only the unmethylated strand of hemimethylated GATC sites that are present immediately after passage of a DNA replication fork. The sensitivity of MutH endonuclease to methylation results in incision of the newly replicated strand. Excision of this strand proceeds through the region of DNA containing the base pair mismatch. This is then followed by resynthesis of the excised strand using the parental strand as a template. The mechanistic steps leading to MutH activation are not clear but have been shown to require ATP hydrolysis (4, 57). Interestingly, only MutS displays an ATP hydrolysis activity; this activity results in the loss of mismatch recognition when MutSdependent mismatch...