Interactions among DNA ligase I, the flap endonuclease and proliferating cell nuclear antigen in the expansion and contraction of CAG repeat tracts in yeast

Among replication mutations that destabilize CAG repeat tracts, mutations of RAD27, encoding the flap endonuclease, and CDC9, encoding DNA ligase I, increase the incidence of repeat tract expansions to the greatest extent. Both enzymes bind to proliferating cell nuclear antigen (PCNA). To understand whether weakening their interactions leads to CAG repeat tract expansions, we have employed alleles named rad27-p and cdc9-p that have orthologous alterations in their respective PCNA interaction peptide (PIP) box. Also, we employed the PCNA allele pol30-90, which has changes within its hydrophobic pocket that interact with the PIP box. All three alleles destabilize a long CAG repeat tract and yield more tract contractions than expansions. Combining rad27-p with cdc9-p increases the expansion frequency above the sum of the numbers recorded in the individual mutants. A similar additive increase in tract expansions occurs in the rad27-p pol30-90 double mutant but not in the cdc9-p pol30-90 double mutant. The frequency of contractions rises in all three double mutants to nearly the same extent. These results suggest that PCNA mediates the entry of the flap endonuclease and DNA ligase I into the process of Okazaki fragment joining, and this ordered entry is necessary to prevent CAG repeat tract expansions.     

The effect of DNA replication mutations on CAG tract stability in yeast.

CAG repeat tracts are unstable in yeast, leading to frequent contractions and infrequent expansions in repeat tract length. To compare CAG repeats to other simple repeats and palindromic sequences, we examined the effect of DNA replication mutations, including alleles of pol alpha, pol delta, pol epsilon, and PCNA (proliferating cell nuclear antigen), on tract stability. Among the polymerase mutations, the pol delta mutation (pol3-14) destabilizes tracts with either CAG or CTG as the lagging strand template. One pol alpha mutation, pol1-1, destabilizes the orientation with CAG as the lagging strand template, but it has little effect on the CTG orientation. In contrast, the pol1-17 mutation has no effect on either orientation. Similarly, mutations in the proofreading functions of pol delta and pol epsilon, as well as a temperature-sensitive pol epsilon mutation, pol2-18, have no effect on tract stability. Three PCNA mutations, pol30-52, pol30-79, and pol30-90, all have drastic effects on tract stability. Of the three, pol30-52 is unique in yielding small tract changes that are indicative of an impairment in mismatch repair. These results show that while CAG repeats are destabilized by many of the same mutations that destabilize other simple repeats, they also have some behaviors that are suggestive of their potential to form hairpin structures.     

In vitro (CTG)*(CAG) expansions and deletions by human cell extracts

The mechanism of disease-associated (CTG)*(CAG) expansion may involve DNA replication slippage, replication direction, Okazaki fragment processing, recombination, or repair. A length-dependent bias for expansions is observed in humans affected by a trinucleotide repeat-associated disease. We developed an assay to test the effect of replication direction on (CTG)*(CAG) instabilities incurred during in vitro (SV40) DNA replication mediated by human cell extracts. This system recapitulates the bias for expansions observed in humans. Replication by HeLa cell extracts generated expansions and deletions that depended upon repeat tract length and the direction of replication. Templates with 79 repeats yielded predominantly expansions (CAG as lagging strand template) or predominantly deletions (CTG as lagging strand template). Templates containing 17 repeats were stable. Thus, replication direction determined the type of mutation. These results provide new insights into the orientation of replication effect upon repeat stability. This system will be useful in determining the contribution of specific human proteins to (CTG)*(CAG) expansions.     

Orientation dependence of trinucleotide CAG repeat instability in Saccharomyces cerevisiae.

To examine the chromosomal stability of repetitions of the trinucleotide CAG, we have cloned CAG repeat tracts onto the 3' end of the Saccharomyces cerevisiae ADE2 gene and placed the appended gene into the ARO2 locus of chromosome VII. Examination of chromosomal DNA from sibling colonies arising from clonal expansion of strains harboring repeat tracts showed that repeat tracts often change in length. Most changes in tract length are decreases, but rare increases also occur. Longer tracts are more unstable than smaller tracts. The most unstable tracts, of 80 to 90 repeats, undergo changes at rates as high as 3 x 10(-2) changes per cell per generation. To examine whether repeat orientation or adjacent sequences alter repeat stability, we constructed strains with repeat tracts in both orientations, either with or without sequences 5' to ADE2 harboring an autonomously replicating sequence (ARS; replication origin). When CAG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively stable regardless of the orientation of ADE2. When CTG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively unstable regardless of the orientation of ADE2. Removal of the ARS as well as other sequences adjacent to the 5' end of ADE2 alters the orientation dependence such that stability now depends on the orientation of ADE2 in the chromosome. These results suggest that the proximity of an ARS or another sequence has a profound effect on repeat stability.     

Mutations in yeast replication proteins that increase CAG/CTG expansions also increase repeat fragility

Expansion of trinucleotide repeats (TNRs) is the causative mutation in several human genetic diseases. Expanded TNR tracts are both unstable (changing in length) and fragile (displaying an increased propensity to break). We have investigated the relationship between fidelity of lagging-strand replication and both stability and fragility of TNRs. We devised a new yeast artificial chromomosme (YAC)-based assay for chromosome breakage to analyze fragility of CAG/CTG tracts in mutants deficient for proteins involved in lagging-strand replication: Fen1/Rad27, an endo/exonuclease involved in Okazaki fragment maturation, the nuclease/helicase Dna2, RNase HI, DNA ligase, polymerase delta, and primase. We found that deletion of RAD27 caused a large increase in breakage of short and long CAG/CTG tracts, and defects in DNA ligase and primase increased breakage of long tracts. We also found a correlation between mutations that increase CAG/CTG tract breakage and those that increase repeat expansion. These results suggest that processes that generate strand breaks, such as faulty Okazaki fragment processing or DNA repair, are an important source of TNR expansions.     

Weak strand displacement activity enables human DNA polymerase beta to expand CAG/CTG triplet repeats at strand breaks

Using synthetic DNA constructs in vitro, we find that human DNA polymerase beta effectively catalyzes CAG/CTG triplet repeat expansions by slippage initiated at nicks or 1-base gaps within short (14 triplet) repeat tracts in DNA duplexes under physiological conditions. In the same constructs, Escherichia coli DNA polymerase I Klenow Fragment exo(-) is much less effective in expanding repeats, because its much stronger strand displacement activity inhibits slippage by enabling rapid extension through two downstream repeats into flanking non-repeat sequence. Polymerase beta expansions of CAG/CTG repeats, observed over a 32-min period at rates of approximately 1 triplet added per min, reveal significant effects of break type (nick versus gap), strand composition (CTG versus CAG), and dNTP substrate concentration, on repeat expansions at strand breaks. At physiological substrate concentrations (1-10 microm of each dNTP), polymerase beta expands triplet repeats with the help of weak strand displacement limited to the two downstream triplet repeats in our constructs. Such weak strand displacement activity in DNA repair at strand breaks may enable short tracts of repeats to be converted into longer, increasingly mutable ones associated with neurological diseases.     

Replication and expansion of trinucleotide repeats in yeast

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) x (CCG)(n) and (CAG)(n) x (CTG)(n) repeats and their propensity to expand in Saccharomyces cerevisiae. Using electrophoretic analysis of replication intermediates, we found that (CGG)(n) x (CCG)(n) repeats significantly attenuate replication fork progression. Replication inhibition for this sequence becomes evident at as few as approximately 10 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) x (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) x (CCG)(25) and (CAG)(25) x (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 approximately 50-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.     

Polar alteration of short tandem repeats (STRs) in mammalian cells

Instability of short tandem repeats (STRs) in DNA during replication is observed in all organisms examined, and is causatively involved in various human diseases. We explore the mechanisms involved in instability by examining length changes occurring during the replication of [(CA)(20)TA](n) and [(CAG)(20)TAG](n), in human cells. We show that the majority of alterations consist of an insertion or deletion of one repeat unit, and base substitutions or length changes involving many repeat units are rare. We also show that length changes of two-tract STRs are biased toward the 3'-end of the repeat tract, in reference to lagging strand synthesis. There are some differences between our observations and previous observations in microbes, e.g. the orientation effect was not observed in this study. The results of this study are discussed in terms of the molecular mechanisms leading to alterations in repeat tracts.     

Hairpin structure-forming propensity of the (CCTG.CAGG) tetranucleotide repeats contributes to the genetic instability associated with myotonic dystrophy type 2

The genetic instabilities of (CCTG.CAGG)(n) tetranucleotide repeats were investigated to evaluate the molecular mechanisms responsible for the massive expansions found in myotonic dystrophy type 2 (DM2) patients. DM2 is caused by an expansion of the repeat from the normal allele of 26 to as many as 11,000 repeats. Genetic expansions and deletions were monitored in an African green monkey kidney cell culture system (COS-7 cells) as a function of the length (30, 114, or 200 repeats), orientation, or proximity of the repeat tracts to the origin (SV40) of replication. As found for CTG.CAG repeats related to DM1, the instabilities were greater for the longer tetranucleotide repeat tracts. Also, the expansions and deletions predominated when cloned in orientation II (CAGG on the leading strand template) rather than I and when cloned proximal rather than distal to the replication origin. Biochemical studies on synthetic d(CAGG)(26) and d(CCTG)(26) as models of unpaired regions of the replication fork revealed that d(CAGG)(26) has a marked propensity to adopt a defined base paired hairpin structure, whereas the complementary d(CCTG)(26) lacks this capacity. The effect of orientation described above differs from all previous results with three triplet repeat sequences (including CTG.CAG), which are also involved in the etiologies of other hereditary neurological diseases. However, similar to the triplet repeat sequences, the ability of one of the two strands to form a more stable folded structure, in our case the CAGG strand, explains this unorthodox "reversed" behavior.     

Absence of MutSβ leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats.     

Gene conversion (recombination) mediates expansions of CTG[middle dot]CAG repeats

Genetic recombination is a robust mechanism for expanding CTG.CAG triplet repeats involved in the etiology of hereditary neurological diseases (Jakupciak, J. P., and Wells, R. D. (1999) J. Biol. Chem. 274, 23468-23479). This two-plasmid recombination system in Escherichia coli with derivatives of pUC19 and pACYC184 was used to investigate the effect of triplet repeat orientation on recombination and extent of expansions; tracts of 36, 50, 80, and 36, 100, and 175 repeats in length, respectively, in all possible permutations of length and in both orientations (relative to the unidirectional replication origins) revealed little or no effect of orientation of expansions. The extent of expansions was generally severalfold the length of the progenitor tract and frequently exceeded the combined length of the two tracts in the cotransformed plasmids. Expansions were much more frequent than deletions. Repeat tracts bearing two G-to-A interruptions (polymorphisms) within either 171- or 219-base pair tracts substantially reduced the expansions compared with uninterrupted repeat tracts of similar lengths. Gene conversion, rather than crossing over, was the recombination mechanism. Prior studies showed that DNA replication, repair, and tandem duplication also mediated genetic instabilities of the triplet repeat sequence. However, gene conversion (recombinational repair) is by far the most powerful expansion mechanism. Thus, we propose that gene conversion is the likely expansion mechanism for myotonic dystrophy, spinocerebellar ataxia type 8, and fragile X syndrome.     

Effects of temperature, Mg2+ concentration and mismatches on triplet-repeat expansion during DNA replication in vitro.

The human genome contains many simple tandem repeats that are widely dispersed and highly polymorphic. At least one group of simple tandem repeats, the DNA trinucleotide repeats, can dramaticallyexpand in size during transmission from one generation to the next to cause disease by a process known as dynamic mutation. We investigated the ability of trinucleotide repeats AAT and CAG to expand in size during DNA replication using a minimal in vitro system composed of the repeat tract, with and without unique flanking sequences, and DNA polymerase. Varying Mg2+concentration and temperature gave dramatic expansions of repeat size during DNA replication in vitro. Expansions of up to 1000-fold were observed. Mismatches partially stabilized the repeat tracts against expansion. Expansions were only detected when the primer was complementary to the repeat tract rather than the flanking sequence. The results imply that cellular environment and whether the growing strand contains a nick or gap are important factors for the expansion process in vivo.     

Triplet repeat expansion generated by DNA slippage is suppressed by human flap endonuclease 1

Human flap endonuclease 1 (h-FEN1) mutations have dramatic effects on repeat instability. Current models for repeat expansion predict that h-FEN1 protein prevents mutations by removing 5'-flaps generated at ends of Okazaki fragments by strand displacement synthesis. The models propose that hairpin formations within flaps containing repeats enable them to escape h-FEN1 cleavage. Friedreich's ataxia is caused by expansion mutations in a d(GAA)n repeat tract. Single-stranded d(GAA)n repeat tracts, however, do not form stable hairpins until the repeat tracts are quite long. Therefore, to understand how d(GAA)n repeat expansions survive h-FEN1 activity, we determined the effects of h-FEN1 on d(GAA)n repeat expansion during replication of a d(TTC)n repeat template. Replication initiated within the repeat tract generated significant expansion that was suppressed by the addition of h-FEN1 at the start of replication. The ability of h-FEN1 to suppress expansion implies that DNA slippage generates a 5'-flap in the nascent strand independent of strand displacement synthesis by an upstream polymerase. Delaying the addition of h-FEN1 to the replication reaction abolished the ability of h-FEN1 ability to suppress d(GAA)n repeat expansion products of all sizes, including sizes unable to hairpin. Use of model substrates demonstrated that h-FEN1 cleaves d(GAA)n 5'-flaps joined to double-stranded nonrepeat sequences but not those joined to double-stranded repeat tracts. The results provide evidence that, given the opportunity, short d(GAA)n repeat expansion products rearrange from 5'-flaps to stable internal loops inside the repeat tract. Long expansion products are predicted to form hairpinned flaps and internal loops. Once formed, these DNA conformations resist h-FEN1. The biological implications of the results are discussed.     

Chromosomal ARS1 has a single leading strand start site

Initiation sites for DNA synthesis in the chromosomal autonomously replicating sequence (ARS)1 of Saccharomyces cerevisiae were detected at the nucleotide level. The transition from discontinuous to continuous synthesis defines the origin of bidirectional replication (OBR), which mapped adjacent to the origin recognition complex binding site. To ascertain which sites represented starts for leading or lagging strands, we characterized DNA replication from ARS1 in a cdc9 (DNA ligase I) mutant, defective for joining Okazaki fragments. Leading strand synthesis in ARS1 initiated at only a single site, the OBR. Thus, replication in S. cerevisiae is not initiated stochastically by choosing one out of multiple possible sites but, rather, is a highly regulated process with one precise start point.     

Effects of sequence on repeat expansion during DNA replication

Small DNA repeat tracts are located throughout the human genome. The tracts are unstable, and expansions of certain repeat sequences cause neuromuscular disease. DNA expansions appear to be associated with lagging-strand DNA synthesis and DNA repair. At some sites of repeat expansion, e.g. the myotonic dystrophy type 2 (DM2) tetranucleotide repeat expansion site, more than one repeat tract with similar sequences lie side by side. Only one of the DM2 repeat tracts, however, is found to expand. Thus, DNA base sequence is a possible factor in repeat tract expansion. Here we determined the expansion potential, during DNA replication by human DNA polymerase β, of several tetranucleotide repeat tracts in which the repeat units varied by one or more bases. The results show that subtle changes, such as switching T for C in a tetranucleotide repeat, can have dramatic consequences on the ability of the nascent-strand repeat tract to expand during DNA replication. We also determined the relative stabilities of self-annealed 100mer repeats by melting-curve analysis. The relative stabilities did not correlate with the relative potentials of the analogous repeats for expansion during DNA replication, suggesting that hairpin formation is not required for expansion during DNA replication.     

Long CTG.CAG repeat sequences markedly stimulate intramolecular recombination

Previous studies have shown that homologous recombination is a powerful mechanism for generation of massive instabilities of the myotonic dystrophy CTG.CAG sequences. However, the frequency of recombination between the CTG.CAG tracts has not been studied. Here we performed a systematic study on the frequency of recombination between these sequences using a genetic assay based on an intramolecular plasmid system in Escherichia coli. The rate of intramolecular recombination between long CTG.CAG tracts oriented as direct repeats was extraordinarily high; recombinants were found with a frequency exceeding 12%. Recombination occurred in both RecA(+) and RecA(-) cells but was approximately 2-11 times higher in the recombination proficient strain. Long CTG.CAG tracts recombined approximately 10 times more efficiently than non-repeating control sequences of similar length. The recombination frequency was 60-fold higher for a pair of (CTG.CAG)(165) tracts compared with a pair of (CTG.CAG)(17) sequences. The CTG.CAG sequences in orientation II (CTG repeats present on a lagging strand template) recombine approximately 2-4 times more efficiently than tracts of identical length in the opposite orientation relative to the origin of replication. This orientation effect implies the involvement of DNA replication in the intramolecular recombination between CTG.CAG sequences. Thus, long CTG.CAG tracts are hot spots for genetic recombination.     

Rev1 enhances CAG.CTG repeat stability in Saccharomyces cerevisiae

Trinucleotide repeats (TNRs) frequently expand in certain human genetic diseases, often with devastating pathological consequences. TNR expansions require the addition of new DNA; accordingly, molecular models suggest aberrant DNA replication or error-prone repair synthesis as the sources of most instability. Some proteins are currently known that either promote or inhibit TNR mutability. To identify additional proteins that help protect cells against TNR instability, yeast mutants were isolated with higher than normal rates of CAG.CTG tract expansions. Surprisingly, a rev1 mutant was isolated. In contrast to its canonical function in supporting mutagenesis, we found that Rev1 reduces rates of CAG.CTG repeat expansions and contractions, as judged by the behavior of the rev1 mutant. The rev1 mutator phenotype was specific for TNRs with hairpin forming capacity. Mutations in REV3 or REV7, encoding the subunits of DNA polymerase zeta (pol zeta), did not affect expansion rates in REV1 or rev1 strains. A rev1 point mutant lacking dCMP transferase activity was normal for TNR instability, whereas the rev1-1 allele that interferes with BRCT domain function was as defective as a rev1 null mutant. In summary, these results indicate that yeast Rev1 reduces mutability of CAG.CTG tracts in a manner dependent on BRCT domain function but independent of dCMP transferase activity and of pol zeta.     

Rad9-mediated checkpoint activation is responsible for elevated expansions of GAA repeats in CST-deficient yeast

Large-scale expansion of (GAA)_n repeats in the first intron of the FXN gene is responsible for the severe neurodegenerative disease, Friedreich’s ataxia in humans. We have previously conducted an unbiased genetic screen for GAA repeat instability in a yeast experimental system. The majority of genes that came from this screen encoded the components of DNA replication machinery, strongly implying that replication irregularities are at the heart of GAA repeat expansions. This screen, however, also produced two unexpected hits: members of the CST complex, CDC13 and TEN1 genes, which are required for telomere maintenance. To understand how the CST complex could affect intra-chromosomal GAA repeats, we studied the well-characterized temperature-sensitive cdc13-1 mutation and its effects on GAA repeat instability in yeast. We found that in-line with the screen results, this mutation leads to ∼10-fold increase in the rate of large-scale expansions of the (GAA)₁₀₀ repeat at semi-permissive temperature. Unexpectedly, the hyper-expansion phenotype of the cdc13-1 mutant largely depends on activation of the G2/M checkpoint, as deletions of individual genes RAD9, MEC1, RAD53, and EXO1 belonging to this pathway rescued the increased GAA expansions. Furthermore, the hyper-expansion phenotype of the cdc13-1 mutant depended on the subunit of DNA polymerase δ, Pol32. We hypothesize, therefore, that increased repeat expansions in the cdc13-1 mutant happen during post-replicative repair of nicks or small gaps within repetitive tracts during the G2 phase of the cell cycle upon activation of the G2/M checkpoint.     

Haploinsufficiency of yeast FEN1 causes instability of expanded CAG/CTG tracts in a length-dependent manner

Trinucleotide repeat diseases, such as Huntington's disease, are caused by the expansion of trinucleotide repeats above a threshold of about 35 repeats. Once expanded, the repeats are unstable and tend to expand further both in somatic cells and during transmission, resulting in a more severe disease phenotype. Flap endonuclease 1 (Fen1), has an endonuclease activity specific for 5' flap structures and is involved in Okazaki fragment processing and base excision repair. Fen1 also plays an important role in preventing instability of CAG/CTG trinucleotide repeat sequences, as the expansion frequency of CAG/CTG repeats is increased in FEN1 mutants in vitro and in yeast cells defective for the yeast homolog, RAD27. Here we have tested whether one copy of yeast FEN1 is enough to maintain CAG/CTG tract stability in diploid yeast cells. We found that CAG/CTG repeats are stable in RAD27 +/- cells if the tract is 70 repeats long and exhibit a slightly increased expansion frequency if the tract is 85 or 130 repeats long. However for CAG-155 tracts, the repeat expansion frequency in RAD27 +/- cells is significantly higher than in RAD27 +/+ cells. This data indicates that cells containing longer CAG/CTG repeats need more Fen1 protein to maintain tract stability and that maintenance of long CAG/CTG repeats is particularly sensitive to Fen1 levels. Our results may explain the relatively small effects seen in the Huntington's disease (HD) FEN1 +/- heterozygous mice and myotonic dystrophy type 1 (DM1) FEN1 +/- heterozygous mice, and suggest that inefficient flap processing by Fen1 could play a role in the continued expansions seen in humans with trinucleotide repeat expansion diseases.     

Detection of mosaicism in lymphocytes of parents of free trisomy 21 offspring

Genetic selection assays were developed to measure rates of deletion of one or more (CAG).(CTG) repeats, or an entire repeat tract, in Escherichia coli. In-frame insertions of >or=25 repeats in the chloramphenicol acetyltransferase (CAT) gene of pBR325 resulted in a chloramphenicol-sensitive (Cm(s)) phenotype. When (CAG)25 comprised the leading template strand, deletion of one or more repeats resulted in a chloramphenicol resistant (Cm(r)) phenotype at a rate of 4 x 10(-2) revertants per cell per generation. The mutation rates for plasmids containing (CAG)43 or (CAG)79 decreased significantly. When (CTG)n comprised the leading template strand the Cm(r) mutation rates were 100-1000 lower than for the opposite orientation. As an initial application of this assay, the effects of mutations influencing mismatch repair and recombination were examined. The methyl directed mismatch repair system increased repeat stability only when (CTG)n comprised the leading template strand. Replication errors made with the opposite repeat orientation were apparently not recognized. For the (CAG)n leading strand orientation, mutation rates were reduced as much as 3000-fold in a recA- strain. In a second assay, out-of-frame mutation inserts underwent complete deletion at rates ranging from about 5 x 10(-9) to 1 x 10(-7) per cell per generation. These assays allow careful quantitation of triplet repeat instability in E. coli and provide a way to examine the effects of mutations in replication, repair, and recombination on repeat instability.