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Originally published In Press as doi:10.1074/jbc.M109761200 on February 6, 2002

J. Biol. Chem., Vol. 277, Issue 16, 13926-13934, April 19, 2002
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In Vitro (CTG)·(CAG) Expansions and Deletions by Human Cell Extracts*

Gagan B. PanigrahiDagger , John D. ClearyDagger §, and Christopher E. PearsonDagger §||

From the Dagger  Program of Genetics and Genomic Biology, The Hospital for Sick Children, and § Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5A 1X8, Canada

Received for publication, October 9, 2001, and in revised form, February 5, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

The expansion of a (CTG)·(CAG) trinucleotide repeat is the genetic basis for at least 11 hereditary neurological, neurodegenerative, or neuromuscular diseases including myotonic dystrophy type 1 (DM1)1 and Huntington's disease (1). Generally, in the non-affected population the length of the repeat tracts range from 5 to 24 repeat units. Although they are polymorphic, these lengths are stably transmitted. Expanded lengths of 25-35 or 34-90 repeats can be genetically unstable. Depending upon the gene, these lengths can lead to disease or be asymptomatic and are termed intermediate-, pre-, or proto-mutation alleles. In DM1 these pre-mutation lengths can expand to disease-associated lengths of ~100 up to thousands of repeats, which exhibit a high degree of genetic instability. The strong dependence of the probability of mutation upon the number of repeats has been termed "dynamic mutation" (2) which describes the increased likelihood for the product of an expansion mutation to undergo a subsequent mutation relative to the precursor (shorter) allele. The increased instability of longer lengths is thought to be due to an increased ability to form mutagenic DNA structures (3, 4). The mechanism of repeat expansion is not known. DNA replication slippage (2), replication direction (5), pausing or blockage of replication fork progression (6), Okazaki fragment processing (7-10), mismatch repair (11, 12), and recombination (13) have been proposed to contribute to repeat expansion.

A role for DNA replication in the process of instability was initially provided by bacterial (5, 6) and yeast (7, 11, 14, 15) models of trinucleotide repeat instability, which implicate the direction of replication through the repeat tract. In these organisms, repeat deletions predominate regardless of the direction of replication. However, the frequency of deletions is greater when the CTG strand, rather than the CAG strand, is the template for lagging strand synthesis (yeast and bacteria) (5). These replication direction effects on repeat stability may be caused by replication blockage (6), which is also sensitive in bacteria, to the direction of replication. None of the assay systems in lower organisms recapitulate the expansion-biased instability observed in humans.

Varying degrees of CTG/CAG instability have been observed between different transgenic mouse models (16-19). The cause for this variable instability is thought to be attributed to cis-elements flanking the repeat tract. One possible cis-element is the location of the replication origin, the site of replication initiation. The location of the most proximal replication origin relative to the repeat tract would determine the direction of replication progression through the repeat. The effect of replication direction by human or mouse proteins on trinucleotide repeat instability is unknown.

To understand CTG/CAG instability mediated by human proteins, we established an assay to measure repeat length changes following SV40 DNA replication. A detailed understanding of primate replication fork dynamics has been gained through the SV40 viral DNA replication system (20, 21). Each replication fork consists of a continuous leading strand and a discontinuous lagging strand, which is composed of multiple Okazaki fragments each about 135-145 nucleotides long (22). Replication of a template with 79 repeats that used the CAG strand as the lagging strand template yielded a bias for expansions. Replication of a template that differed only in the direction of replication, CTG as the lagging strand template, yielded a bias for deletions. These results support a role for replication direction in CTG/CAG repeat instability such that the replication direction determined whether expansion or deletion mutations were incurred.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

Replication Templates-- The (CTG)·(CAG) replication templates were designed as follows. Genomic clones of DM1 containing EcoRI/HindIII (CTG)n fragments were subcloned into pBluescript KSII+ (Stratagene). All clones had the repeat in the stable orientation relative to the uni-directional bacterial ColE1 origin of replication (CAG strand as lagging strand template). The length and purity of all (CTG)n repeat tracts were confirmed by sequencing. The SphI-HindIII fragment of the SV40 virus (viral positions 128 and 5171, respectively), which contains the SV40-ori, was PCR-amplified with primers (5'-CTTATCTCTAGAAGCTTTTTGC-3' and 5'-CGCTCTAGATGGTGCATGCATCTC-3') and inserted into pBluescript KSII+ using the XbaI site in the primers (underlined). This 219-nucleotide fragment contains the SV40 origin of bidirectional replication (viral position 5210/5211) (23, 24). The SV40-ori was cloned as a blunted XbaI fragment into either the HindIII or EcoRI sites of pBluescript KSII+ placing the SV40 origin of bidirectional replication 103 and 98 bp 5' and 3' of the CAG repeat, respectively (see Fig. 1). For each template the orientation of the SV40-ori was confirmed by restriction analysis. Large scale plasmid preparations were prepared from dam+ Escherichia coli cells as described previously (4). Briefly, cells were harvested and lysed with lysozyme (Invitrogen) and a detergent solution of 1% Brij 58 (Sigma) and 0.4% deoxycholate (Sigma). Plasmids were treated with RNase A and T1 (Sigma), phenol-extracted, and purified twice by cesium chloride/ethidium bromide centrifugation.

Cell Lines and Cell Extract Preparation-- HeLa S3 cells propagated in suspension cultures were purchased from the National Cell Culture Center, National Center for Research Resources, National Institutes of Health (Bethesda). Cell extracts were prepared as described previously (25, 26). Protein concentration was determined by Bio-Rad Protein Assay kit and used as noted in the text.

In Vitro Replication-- Reactions were performed as described (25, 26). DNA templates (150 ng) were replicated in reactions containing the following final concentrations: dATP, dGTP, dTTP, and dCTP, 100 µM each; GTP, UTP, and CTP, 200 µM each; ATP, 4 mM; creatine phosphate (Roche Molecular Biochemicals), 40 mM; creatine kinase (Roche Molecular Biochemicals), 100 µg/ml; 1 µg of SV40 T-antigen (Chimerx); and 30-µl cell extracts (in the present study we have used 1.06 µg/µl = 32 µg/reaction, but we have found that 1-6 µg/µl are sufficient to initiate and complete in vitro replication). For direct analysis of the replication products, 0.033 µM = 0.099 Ci of [alpha -32P]dCTP (3000 Ci/mmol, PerkinElmer Life Sciences) was included in each 50-µl reaction. Reactions were incubated for 4 h at 37 °C and terminated with 50 µl of stop solution (2 µg/µl proteinase K, 2% SDS, and 50 mM EDTA, pH 8.0) with further incubation for 30 min at 37 °C. Carrier tRNA (15 µg) was added, and protein was extracted twice with phenol/chloroform and chloroform. Replication products were ethanol-precipitated and resuspended in water for further analysis.

Direct Analysis of the Replication Products-- Inter-template replication efficiencies (Fig. 2) were determined as follows. Radioactive replication products were purified, linearized with BamHI, and treated with DpnI or MboI. Equal quantities of reaction products digested with BamHI only or BamHI + DpnI or BamHI + MboI were resolved by electrophoresis on a 15-cm 1% agarose gel. The gel was run for 16 h at 4 V/cm in TBE buffer, dried, and exposed to Kodak film.

Mutation Analysis, the STRIP Assay-- Mutation analysis was performed using the Stability of Trinucleotide Repeats by Individual Product analysis (STRIP assay) as outlined in Fig. 3. In vitro replication products were digested with 10 units of DpnI (New England Biolabs) to eliminate un-replicated parental templates. DpnI-digested material was transformed into E. coli DH5alpha -mcr, and only the DpnI-resistant material gave rise to colonies (27). Individual bacterial colonies, each representing an individual product of primate replication, were picked and cultured for a limited growth period (maximum of 6 h, 4-6 generations). During colony propagation bacterial contribution was minimized for all clones by 1) having the (CTG)·(CAG) repeat in the stable orientation relative to the uni-directional bacterial ColE1 origin of replication; 2) the limited colony growth time (<= 6 h); and 3) bacterial strain selection. Of the >15 bacterial strains tested we selected DH5alpha -mcr (Invitrogen) for analysis of the SV40 replication products, due to its ability to stably propagate (CTG)n-containing plasmids. Through the use of these stringent conditions we were capable of stably maintaining as many as 255 CTG repeats in bacteria (4, 28). Miniprep DNA was analyzed for changes in repeat length by analysis of the repeat-containing fragment on high resolution 4% polyacrylamide gels (29) (Fig. 4). Repeat lengths were classed into one of three categories: "less than 79 repeats," "79 repeats," and "greater than 79 repeats" (Fig. 5).

To focus upon mutation events derived from in vitro replication by human proteins, the background length heterogeneity within the parental template preparation and that derived through bacterial miniprep culture was determined. Background length heterogeneity for each template was determined by direct bacterial transformation of the same template DNA used for the in vitro replication reactions, repeat length analysis, and length categorization of single colonies (Fig. 5). Because the degree and distribution of length heterogeneity may vary between different bacterial preparations of a particular template, it is critical that the same template preparation used for the in vitro replication reactions be used for the bacterial background correction. The significance of differences between repeat length distributions of in vitro replication products and parental template preparations was determined using the chi 2 or Fisher's exact test. For mutation spectra that were significantly different from bacterial background (p < 0.05), the frequency of expansions or deletions generated by primate replication was corrected by subtracting the bacterial background frequency (Table I). We report only increased frequencies of events, i.e. positive differences between the replicated and parental distributions. Because the frequency of events are proportions, an increased frequency of molecules in either the "larger" or "shorter" length categories is necessarily coupled with a decreased frequency in at least one other length category. Following subtraction of the background, those primate-mediated events that were increased resulted in a positive number, reflecting the increased events mediated by in vitro replication. Those length categories that were under-represented relative to the parental material reflect a reduced number of primate-mediated events maintaining or leading to that size category. After subtracting the background from such reduced events resulted in a negative number reported as "0a" thus indicating that the number of replicated molecules in that length category was reduced relative to the parental material (or "no increased events"). For details please see "Results," Fig. 5, and Table I. This rigorous approach ensured that only the human cell extract-mediated expansion or deletion events were reported (Table I) and that these were not a function of the distribution of repeat arrays in the starting material.

Analysis of Sequence and Magnitude of Repeat Length Changes-- The magnitudes of repeat length changes (Fig. 6) were determined by electrophoretic sizing of the repeat containing fragments on 4% polyacrylamide gels (29) relative to the starting length material and a known set of size markers (a representative example is shown in Fig. 4a).

To confirm that the changes observed were due to changes in the repeat tract length, samples were electrotransfered (1.5 mA/cm2, 45 min, Panther Semidry Electroblotter (HEP-1), Owl Separation Systems) to a Biodyne B membrane (0.45 µm, Pall Corp.). The membrane was probed with a 32P-labeled (CTG)15 oligonucleotide and exposed to film (a representative example is shown in Fig. 4b). Additionally, all expansion products and selected products of deletion events were analyzed by sequencing.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

To investigate the role of DNA replication direction on repeat instability, we took advantage of the well studied SV40 replication system which, in addition to being a model of the primate DNA replication fork (30), has been used to study replication fidelity (26), DNA mutagenesis (31), and DNA repair (32). To test repeat stability in this in vitro DNA replication system, we used templates containing both (CTG)·(CAG) repeats and the SV40-ori and replicated them using cell extracts of human cells and SV40 T-antigen. Replication templates contained either 17 or 79 (CTG)·(CAG) repeats, lengths that are typical of the genetically stable non-affected and genetically unstable permutation/diseased individuals, respectively. The location of the SV40-ori determined the direction of replication fork progression through the repeats and thus which strand (CTG or CAG) served as a lagging strand template (Fig. 1). For pDM79E, the CTG strand served as the lagging strand template, whereas for pDM79H, the CAG strand served as a lagging strand template. For this study we used extracts of the human cell line HeLa, which are functional in in vitro replication and various in vitro repair and recombination activities.


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  		Fig. 1.   Schematic diagram of replication templates. a, the pDM79 circular plasmid contains the (CTG)n·(CAG)n (n = 79) in the stable orientation relative to the bacterial unidirectional ColE1 origin of replication. pDM79E and pDM79H were created by inserting the SV40-ori containing fragment into the EcoRI and HindIII sites, respectively. The sites of SV40-ori insertion are shown as EcoRI and HindIII. b, location of SV40-ori determines the replication direction and which strand will serve as the leading or lagging strand template. The direction of replication fork progression for pDM79E initiates 3' of the CAG and pDM79H initiates 5' of the CAG tract. c, schematic of the templates showing the distance between SV40-Origin of Bidirectional Replication (viral positions 5210/5211) (24) (represented as bubble) and repeat tract. An identical set of templates harboring 17 repeats and the SV40-ori were generated. The pKN16 template contains the SV40-ori but no repeat tracts.

Replication Efficiency and Direct Analysis of in Vitro Replication Products-- To determine whether there were template-specific differences in the efficiency of replication, each template was replicated in vitro with HeLa cell extracts, and the amount of completely replicated and re-replicated material was compared. Reaction products were linearized (BamHI) and then digested with either of the methyl-sensitive enzymes DpnI or MboI (Fig. 2). DNAs resistant to DpnI represent products of at least one complete round of replication. DNAs digested by MboI represent products of more than one complete round of replication. As judged by the DpnI-resistant material, there did not appear to be any inter-template differences in replication efficiency (Fig. 2, compare lanes 2, 4, and 6). The similar incorporation pattern of the DpnI digestion products between the templates indicates that there was no difference in the ability to initiate and extend replication through the repeat tract from either direction. The similar sensitivities to MboI (not shown) revealed that there were no differences in their abilities to re-initiate and complete subsequent rounds of replication. Thus, the replication efficiencies of the repeat-containing templates pDM79E and pDM79H were similar to each other and to that of pKN16, which contained no repeats (Fig. 2, compare lanes 2, 4, and 6). We conclude that there were no repeat-specific or replication direction-specific differences in the efficiencies of in vitro replication.


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  		Fig. 2.   Determining the efficiency of template replication. Different SV40-ori templates containing repeats (pDM79H and pDM79E) or no repeats (pKN16) were replicated in vitro by HeLa cell extracts (32 µg of protein/reaction). Purified products were linearized with BamHI, and an equal portion of this material was also digested with both BamHI and DpnI. The digestion products were electrophoresed on a 1% agarose gel to resolve the completely replicated and un-replicated material. An autoradiogram of the dried gel is shown. The DpnI-resistant material in lanes 2, 4, and 6 represents the products of at least one complete round of replication.

Mutation Analysis, the STRIP Assay-- To determine the effect of in vitro replication by human proteins on repeat stability, we developed a sensitive assay to measure CTG tract stability of pDM79H and pDM79E following in vitro replication. Repeat stability was measured using the STRIP assay as outlined in Fig. 3 (see also "Experimental Procedures"). Products of in vitro replication were isolated and digested with DpnI to eliminate un-replicated parental DNAs. Only DpnI-resistant material (the product of in vitro replication) is capable of transforming bacteria (27). Individual bacterial colonies therefore represent individual products of in vitro replication. Plasmids isolated from the individual bacterial colonies were restriction-digested to release the repeat-containing fragment. Following analysis on high resolution polyacrylamide gels, expansion or deletion events were scored (a representative example is shown in Fig. 4a). Expansion events were observed as distinct bands migrating slower than the starting length (CTG)79 band, whereas deletion events migrated faster. To confirm that these events were due to changes in the repeat tract, samples were electrotransfered and probed with 32P-(CTG)15 oligonucleotide (Fig. 4b). Furthermore, individual mutation products were sequenced, confirming that changes were limited to increases or decreases of integral numbers of repeat units.


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  		Fig. 3.   Outline of the STRIP analysis. Replication templates containing SV40-ori and trinucleotide repeats were replicated in vitro by HeLa cell extracts and T-antigen. Replication products were purified and digested with DpnI. DpnI-resistant material (gray molecules) represents products of complete replication by HeLa cell extract. DpnI digestion eliminated unreplicated (bold molecules) or partially replicated material (gray/bold molecules). Reactions were transformed into E. coli, and only the DpnI-resistant material yielded colonies, with each colony representing an individual in vitro replication product (27). Plasmid DNAs were isolated, the repeat-containing fragment released by restriction digestion, and analyzed for length on high resolution polyacrylamide gels. For an example, see Fig. 4. For details see "Experimental Procedures" and "Results."


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  		Fig. 4.   Repeat length analysis of the replication products. Primate replication products were processed as described in Fig. 3. Individual bacterial colonies each representing individual in vitro replication products were picked, plasmid-isolated, and restriction-digested to release the repeat-containing fragment. a, DNAs were resolved on 4% polyacrylamide gels and products visualized by ethidium bromide. Shown are products with no change (lanes 1-3), deletions (lanes 4-6), and expansions (lanes 7-9). Lane 10 shows the intact repeat length of the starting plasmid digested with restriction enzymes. b, shows the same gel transferred to a nylon membrane and hybridized with [32P] (CTG)15 probe.

To focus upon primate replication-mediated mutation events, it was necessary to correct for both the low levels of repeat length heterogeneity present in the template preparation (28) and the limited heterogeneity produced during bacterial culture ("Experimental Procedures"). In humans and in model systems trinucleotide repeat tract lengths are best described as a distribution. Background levels for the pDM79E and pDM79H templates were determined by direct bacterial transformation of the same starting template DNA used for in vitro replication mediated by human cell extracts. Individual molecules (colonies) were analyzed for repeat length (as outlined in Fig. 4), and these were classed into one of three categories: "less than 79 repeats," "79 repeats," and "greater than 79 repeats" (Fig. 5, see open bars). The molecules in each of these categories could experience primate replication-mediated expansion or deletion events. For those primate-mediated replication products whose length distribution frequencies were significantly different (p < 0.05) from the background length heterogeneity, the primate contribution was corrected for background by subtraction (see below).


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  		Fig. 5.   Distribution of repeat lengths before and after replication by human cell extracts. The actual number of molecules observed with different length distributions are shown over the total number of molecules analyzed. Exact numbers are indicated. The height (y axis) of the bars is the % of molecules observed. a, the distribution of repeats before (background, open bars) and after in vitro replication by HeLa cell extracts (filled bars) for plasmid pDM79H. Note the shift in distribution with more molecules having "greater than" or equal to "79 repeats" and the accompanying decrease in molecules "less than 79 repeats." b, the distribution of repeats before (background, open bars) and after in vitro replication by HeLa cell extracts (filled bars) for plasmid pDM79E. Note the shift in distribution with more molecules having "less than 79 repeats" and the accompanying decrease in molecules having "more than 79 repeats." The statistical significance of the results is shown as determined by using chi 2 or Fisher's exact test for pDM79H or pDM79E, respectively. Mutation frequencies corrected for background are presented in Table I.

Effect of Replication Direction and Tract Length on Trinucleotide Repeat Instability-- The direction of in vitro replication had a significant effect upon the stability of the repeat tract. The effect of in vitro replication direction on repeat stability was determined by measuring CTG tract stability of pDM79H and pDM79E following in vitro replication. The length distribution of the replication products for each template was determined by STRIP analysis (as outlined in Fig. 3), and results are presented in Fig. 5 (see filled bars). For pDM79H the frequency of observed HeLa replication products with repeat lengths "larger than 79 repeats" was higher than that present in the starting material (Fig. 5, pDM79H compare filled bars with hollow bars). Furthermore, the frequency of observed HeLa replication products with lengths "less than 79 repeats" or containing the intact length of "79 repeats" was less than that contained in the parental template preparation. This supports an overall expansion bias for pDM79H. In contrast for pDM79E the frequency of HeLa replication products with repeat lengths "less than 79 repeats" was higher than that present in the starting material (Fig. 5, pDM79E compare filled bars with open bars). Furthermore the frequency of observed HeLa replication products with lengths "larger than 79 repeats" or containing the intact length of "79 repeats" was less than that present in the parental template preparation. Together these results suggest that altered repeat lengths of pDM79H in vitro replication products were predominantly the result of expansion events, whereas the altered lengths of pDM79E in vitro replication products were predominantly the result of deletion events. The significance of these findings was determined using chi 2 or Fisher's exact test comparing the trichotomized length distributions of the in vitro replication products with that of the parental template (background). The length distributions of in vitro replicated pDM79H and pDM79E were significantly different from their respective background length heterogeneities (p = 0.047 and p = 0.005, respectively). Thus, in vitro replication of pDM79H and pDM79E by human cell extracts resulted in a bias for expansions and a bias for deletions, respectively. The HeLa-mediated expansion and deletion frequencies reported in Table I were corrected by subtracting the background frequencies. Thus, pDM79H displayed 7% HeLa-mediated expansions, which is the significant increase of the greater than 79 repeat category (7% = (11/73) - (14/168) × 100). Because these are proportions, an increased percentage of molecules in one category is necessarily coupled with a decrease in the percentage of molecules in at least one other category. In the case of pDM79H the increased frequency of expansions is coupled with a decreased frequency of deletions (Fig. 5, compare filled bars with open bars). Hence, the reduced frequency of events or the lack of increased frequency of events following primate replication is reported as 0a in Table I. Similar correction revealed that pDM79E displayed 17% HeLa-mediated deletions (17% = (30/76) - (31/141) × 100) and no significant increase of expansions. We conclude that direction of in vitro replication of a (CTG)79·(CAG)79 tract by human cell extracts leads to a bias for expansions when the CAG is the lagging strand template and a bias for deletions when the CTG is the lagging strand template. The change of mutation type from expansion to deletion between the two templates indicated that the direction of in vitro replication by human cell extract had a profound effect on repeat instability.

                              
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Table I
Corrected frequencies following in vitro replication
Expansion and deletion frequencies mediated by in vitro replication by HeLa extracts were corrected for background by subtracting the proportion of parental template molecules from the observed frequency of replication products with greater than 79 or less than 79 repeats (see under "Experimental Procedures" and "Results"). Repeat length distributions of replication products that were not significantly different from their corresponding parental repeat length heterogeneity (background) (using the chi 2 or Fisher's exact test, p > 0.05) were classed as stable or having "0" primate-induced events. For templates with statistically significant differences between the repeat length distribution of in vitro replicated and template background (using the chi 2 or Fisher's exact test, p > 0.05) was corrected by subtraction. Within a given length category the proportion of template molecules was subtracted from the proportion of replicated molecules. For those events that increased following replication a positive value was obtained, whereas for those events that had decreased occurrences a negative value was obtained (no increased events) and is reported as 0a. NA, not applicable.

To determine the effect of repeat length on repeat instability, we used a similar set of replication templates containing only 17 repeats, a length that is typically stable in humans. Templates were replicated in vitro, and repeat stability was analyzed by the STRIP assay. Neither the parental templates or in vitro replication products of pDM17H or pDM17E template revealed any repeat length changes; hence, they were stable (Table I) indicating an association between repeat length and instability.

Magnitude of Repeats Gained or Lost Does Not Vary with the Direction of Replication-- It was of interest to know if the magnitude of repeats gained or lost following in vitro replication varied depending upon replication direction. The repeat length of each of the individual parental molecules (open bars) and of each in vitro replicated molecule (filled bars) was determined, batched according to repeat length, and scaled according to the percentage of molecules within that length range. In vitro replication of pDM79H by HeLa cell extracts resulted in predominantly expansions, with a mean of +7 repeats and a range of +1 to +28 repeats (Fig. 6, filled bars). Increases of +28 were greater than the largest expansion of +8 repeats incurred by bacterial replication of the same template (compare open bars with filled bars). In vitro replication of pDM79E resulted in predominantly deletions with mean of -22 repeats and a range of -2 to -55 repeats. Comparison of the repeat lengths of pDM79H in vitro expansion products with the lengths of the parental templates having lengths "longer than 79" revealed a significant trend toward larger repeat lengths for the replicated pDM79H (using a two-sided Mann-Whitney test, p = 0.018). Comparison of the repeat lengths of pDM79E in vitro deletion products with the lengths of the parental templates having lengths "shorter than 79" did not reveal a significant difference in repeat lengths for the replicated pDM79E (p = 0.845). However, for this template comparison of the lengths of the replicated molecules with the template molecules in the larger than 79 repeats category revealed a significant (p = 0.027) reduction for the replication products. We interpret this as an indication of intra-category (larger than 79 repeats) deletion events. These analyses reveal that the magnitude of repeats gained during in vitro expansion are significantly larger, but the magnitudes of repeats lost during in vitro replication were not. Thus, the magnitudes of repeats gained or lost and the mutation frequencies are consistent with a bias for expansions and a bias for deletions following primate replication of pDM79H and pDM79E, respectively.


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  		Fig. 6.   Magnitude of repeat length change, pDM79H and pDM79E. In vitro replicated products were processed as described in Fig. 3. Following resolution on 4% polyacrylamide gels, the repeat-containing fragments of individual replication products were sized ("Experimental Procedures"). Repeat length changes were batched in size ranges of 10 repeats above and below the starting length of 79 repeats (x axis). To facilitate comparison both the percentage of molecules within each class (y axis) and the actual numbers observed and analyzed (below x axis) are represented. Both the in vitro replicated distribution (solid bars) and parental template background distribution (open bars) are represented.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

We have developed an in vitro replication assay that uses human cell extracts to investigate the mechanism of (CTG)·(CAG) repeat instability. This assay has the capability to test the possible contribution of cis-elements and trans-acting factors to repeat instabilities incurred during in vitro DNA replication by human proteins. Herein we have tested two cis-elements including repeat tract length and replication direction. Both tract length and replication direction were important determinants of repeat instability.

Effect of Repeat Tract Length and Replication Direction-- In humans the number of (CTG)·(CAG) repeats in a tract is a major determinant of its genetic instability (2). A role for replication direction in (CTG)·(CAG) instability has been observed in both bacteria (5) and yeast (14). In our primate system, both tract length and replication direction affected repeat stability. Instability was dependent upon repeat tract length, only templates with expanded (CTG)79 yielded detectable levels of instability, whereas shorter, (CTG)17, templates were stably replicated. Unlike the bacterial and yeast systems, both of which overwhelmingly yield deletions regardless of replication direction (5, 8, 14), our primate system suggested that the direction of replication determined the type of mutation (expansions versus deletions). Predominantly expansions were detected for the pDM79H template, in which the CAG strand served as the lagging strand template, whereas predominantly deletions were detected for the pDM79E template, in which the CTG strand served as the lagging strand template. Therefore our in vitro replication system recapitulates the bias for expansion observed in patients affected with a trinucleotide repeat-associated disease.

It is unclear how the direction of primate replication fork progression through the repeat determines whether expansions or deletions follow. The cause for the replication direction effect may occur at one or several of three possible steps including the formation of mutagenic intermediates, their correct or escaped repair, and their possible aberrant repair/processing (Fig. 7). First, the propensity to form the mutagenic intermediates may vary depending upon which repeat strands serve as the leading and lagging strand templates (Fig. 7, top). Such intermediates may occur on either or both the leading or lagging branches of the replication fork, and possibly even ahead of the fork. Second, the ability of certain mutagenic intermediates to be correctly repaired or to escape repair may vary depending upon which repeat strand is slipped out (Fig. 7, middle and lower left). The third area is aberrant repair (or processing) of the mutagenic intermediates that would lead to products having repeat lengths different from the parental template (Fig. 7, lower right). Mutation fixation by aberrant processing requires enzymatic activity, whereas escaped repair is a lack of activity. Below we have briefly discussed our results and how they reflect upon each of these steps.


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  		Fig. 7.   Possible mutagenic intermediates and mutation pathways. Different directions of replication through the repeat may cause the formation of different mutagenic intermediates. The propensity to form specific mutagenic intermediates may be mediated by which strand serves as the leading or lagging strand template (top). Depending upon the direction of replication the types of expansion or deletion mutagenic intermediates may have an excess of either CTG or CAG repeats in either the nascent or template strand (middle). Mutagenic structures may even be formed ahead of the replication fork (top). Structural features of specific mutagenic intermediates may determine whether they are correctly repaired or escape repair or are aberrantly repaired (bottom).

Propensity to Form Mutagenic Intermediates-- A differential propensity to form mutagenic intermediates when replicating from one or the other direction may explain the replication direction effect we observed (Fig. 7, top). In bacterial and yeast models it was originally suggested (5, 14) that the differential ability to form hairpins by the CTG relative to the CAG repeat (3) could explain the orientation effect on the propensity to incur deletions. An extension of this model may explain the replication direction-dependent expansion or deletions we have observed following in vitro replication by human proteins. If one assumes that mutagenic intermediates form on only the lagging branch of a replication fork (Fig. 7, top) expansion intermediates of pDM79H would contain slipped out CTG repeats, whereas deletion intermediates would contain slipped out CAG repeats. In contrast expansion intermediates of pDM79E would contain slipped out CAG repeats and deletions intermediates would contain slipped out CTG repeats. The increased ability to form stable slipped out CTG repeats may favor the expansion bias and deletion bias displayed by pDM79H and pDM79E, respectively. (A similar proposal could be made if one assumed that mutagenic intermediates formed only on the leading branch of the replication fork.) The differential ability to form one mutagenic intermediate over another may not be limited to the biophysical structure of the DNA but may be mediated by certain DNA structure-specific proteins. We have shown that the bacterial single strand-binding protein and the mismatch repair protein hMSH2 binds more readily to slipped out CAG repeats than to slipped out CTG repeats (33).2 A DNA structure-specific protein may favor or disfavor the formation of some but not all slipped structures.

It has been suggested that replication fork pausing in the repeat tracts, which is sensitive to replication direction, may be related to the ability to form mutagenic intermediates (5, 6). We did not detect a difference in the replication efficiency between pDM79H and pDM79E, both were similar to each other and to that of a template void of repeat sequences. The similar efficiency of replication in either direction and the similar incorporation patterns of the incompletely replicated DpnI-sensitive products did not reveal a differential ability to initiate, elongate, or complete replication. Whereas these findings argue against a replication pausing type of mechanism, it is possible that there is a differential ability to form mutagenic intermediates that does not involve pausing of replication progression.

Correct or Escaped Repair of Mutagenic Intermediates-- The differential ability of certain mutagenic intermediates to be correctly repaired or to escape repair may explain the replication direction effect. The structural features of slipped structures, including the slip-out and the slip-out junction may critically determine whether they are repaired or not. Mutagenic intermediates containing slipped out CAG repeats may be correctly repaired, whereas those with slipped out CTG repeats may escape or be protected from repair (Fig. 7, bottom). We have shown that the biophysical structure of slipped out CAG slipped-intermediate heteroduplex and its three-way junction is considerably different from the structure of the sister slipped out CTG slipped-intermediate heteroduplex and its three-way junction (33).2 Importantly, the slipped out CAG repeats predominantly assumed a random coil single-stranded configuration, whereas the slipped out CTG predominantly assumed a hairpin structure. Furthermore, the slipped out CAG repeat was preferentially bound by single strand-binding protein. Preferential binding of one strand over the other may lead to differences in how the two slip-outs are processed, which may explain the direction of replication effect on repeat instability. Preferential binding of one intermediate over another by a DNA structure-specific protein such as a repair or replication protein (33)2 may facilitate or protect against correct repair.

Aberrant Processing of Mutagenic Intermediates-- The differential ability of certain mutagenic intermediates to be aberrantly repaired, processed, or recombined may explain the orientation effect. The structural features of slipped structures, including the slip-out and the slip-out junction, may critically determine whether they are recognized and processed by replication, repair, or recombination proteins. One model of somatic (CTG)·(CAG) instability involves inefficient processing of slipped Okazaki fragments initiated within the repeat tract by the Flap-endonuclease 1, FEN1 (7, 9, 10, 34). Differential processing of slipped out CTG or CAG hairpins, such that one escaped processing by FEN1, might explain the replication direction effect. However, biochemical studies (9) have revealed that neither CTG nor CAG hairpins are processed inefficiently, with little difference between the two. In yeast it has been clearly shown that heteroduplex DNAs with intra-strand hairpins composed of (CTG)10 or (CAG)10 escaped repair (35), and this finding is similar to the inefficient repair of palindromic heterologies in yeast (36). However, trinucleotide repeats may be processed differently in yeast than in human cells. Notably in yeast trinucleotide repeats tend to delete, which is in contrast to the bias for expansions observed in humans. Furthermore, unlike yeast (35), palindromic heteroduplexes are efficiently repaired by mammalian cells (37). Recent results from transgenic mice indicate that MSH2 is required for CTG/CAG instability (19), consistent with hMSH2 binding to slipped structures (33), and surprisingly MSH3 but not MSH6 is required for instability (38). It is of interest to know if human cells or their extracts are capable of metabolizing slipped-strand trinucleotide repeat structures and what role, if any, the slip-out and CTG/CAG three-way junctions may play. We note that the human cell extracts that we have used are competent in in vitro replication (25), mismatch repair (39), excision repair (including repair of psoralens cross-links, pyrimidine dimers, UV damage, and cisplatin adducts) (40), double-strand break repair (41), homologous recombination (42), and triplex-mediated recombination (43).

Regardless of the precise mechanism, our results with human cell extracts clearly present a striking effect of replication direction on repeat instability. Having established this system, which enables genetic and biochemical manipulation, allows us to address which of the three steps (Fig. 7) contribute to the replication direction effect we have observed.

    CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

We have established an in vitro DNA replication system that uses human cell extracts to study trinucleotide repeat instability. This system recapitulates the bias for expansion observed in humans affected with a trinucleotide repeat-associated disease. This assay can be used to study the biochemical, cellular, and genetic factors that critically affect repeat expansions and deletions.

    ACKNOWLEDGEMENTS

We are grateful to our lab members for comments on the manuscript. We thank Drs. Marc Wold and Peter Bullock for technical advice and Dr. Andrew Paterson for assisting with the statistical analysis.

    FOOTNOTES

* This work was supported in part by grants from the Muscular Dystrophy Association (United States) and the Canadian Institutes of Health Research (to C. E. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by Natural Sciences and Engineering Research Council of Canada.

|| Canadian Institutes of Health Research Scholar, a Canadian Genetic Disease Network Scholar, and a Premier's Research Excellence Award Scholar. To whom correspondence should be addressed: Genetics and Genomic Biology, The Hospital for Sick Children, 555 University Ave., Elm Wing, 11-135, Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-8256; Fax: 416-813-4931; E-mail: cepearson@genet.sickkids.on.ca.

Published, JBC Papers in Press, February 6, 2002, DOI 10.1074/jbc.M109761200

2 C. E. Pearson, M. Tam, Y.-H. Wang, S. E. Montgomery, A. Dar, J. D. Cleary, and K. Nichol, submitted for publication.

    ABBREVIATIONS

The abbreviation used is: DM1, myotonic dystrophy type 1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

1. Cummings, C. J., and Zoghbi, H. Y. (2000) Hum. Mol. Genet. 9, 909-916[Abstract/Free Full Text]
2. Richards, R. I., and Sutherland, G. R. (1992) Cell 70, 709-712[CrossRef][Medline] [Order article via Infotrieve]
3. Gacy, A. M., Goellner, G., Juranic, N., Macura, S., and McMurray, C. T. (1995) Cell 81, 533-540[CrossRef][Medline] [Order article via Infotrieve]
4. Pearson, C. E., and Sinden, R. R. (1996) Biochemistry 35, 5041-5053[CrossRef][Medline] [Order article via Infotrieve]
5. Kang, S., Jaworski, A., Ohshima, K., and Wells, R. D. (1995) Nat. Genet. 10, 213-218[Medline] [Order article via Infotrieve]
6. Samadashwily, G. M., Raca, G., and Mirkin, S. M. (1997) Nat. Genet. 17, 298-304[Medline] [Order article via Infotrieve]
7. Freudenreich, C. H., Kantrow, S. M., and Zakian, V. A. (1998) Science 279, 853-856[Abstract/Free Full Text]
8. Schweitzer, J. K., and Livingston, D. M. (1999) Genetics 152, 953-963[Abstract/Free Full Text]
9. Spiro, C., Pelletier, R., Rolfsmeier, M. L., Dixon, M. J., Lahue, R. S., Gupta, G., Park, M. S., Chen, X., Mariappan, S. V., and McMurray, C. T. (1999) Mol. Cell 4, 1079-1085[CrossRef][Medline] [Order article via Infotrieve]
10. Henricksen, L. A., Tom, S., Liu, Y., and Bambara, R. A. (2000) J. Biol. Chem. 275, 16420-16427[Abstract/Free Full Text]
11. Schweitzer, J. K., and Livingston, D. M. (1998) Hum. Mol. Genet. 7, 69-74[Abstract/Free Full Text]
12. Jaworski, A., Rosche, W. A., Gellibolian, R., Kang, S., Shimizu, M., Bowater, R. P., Sinden, R. R., and Wells, R. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11019-11023[Abstract/Free Full Text]
13. Richard, G. F., Goellner, G. M., McMurray, C. T., and Haber, J. E. (2000) EMBO J. 19, 2381-2390[CrossRef][Medline] [Order article via Infotrieve]
14. Freudenreich, C. H., Stavenhagen, J. B., and Zakian, V. A. (1997) Mol. Cell. Biol. 17, 2090-2098[Abstract]
15. Miret, J. J., Pessoa-Brandao, L., and Lahue, R. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12438-12443[Abstract/Free Full Text]
16. Bates, G. P., Mangiarini, L., Mahal, A., and Davies, S. W. (1997) Hum. Mol. Genet. 6, 1633-1637[Abstract/Free Full Text]
17. Gourdon, G., Radvanyi, F., Lia, A. S., Duros, C., Blanche, M., Abitbol, M., Junien, C., and Hofmann-Radvanyi, H. (1997) Nat. Genet. 15, 190-192[CrossRef][Medline] [Order article via Infotrieve]
18. Monckton, D. G., Coolbaugh, M. I., Ashizawa, K. T., Siciliano, M. J., and Caskey, C. T. (1997) Nat. Genet. 15, 193-196[CrossRef][Medline] [Order article via Infotrieve]
19. Manley, K., Shirley, T. L., Flaherty, L., and Messer, A. (1999) Nat. Genet. 23, 471-473[CrossRef][Medline] [Order article via Infotrieve]
20. DePamphilis, M. L., and Wassarman, P. M. (1980) Annu. Rev. Biochem. 49, 627-666[CrossRef][Medline] [Order article via Infotrieve]
21. Waga, S., and Stillman, B. (1998) Annu. Rev. Biochem. 67, 721-751[CrossRef][Medline] [Order article via Infotrieve]
22. Anderson, S., and DePamphilis, M. L. (1979) J. Biol. Chem. 254, 11495-11504[Abstract/Free Full Text]
23. Hay, R. T., Hendrickson, E. A., and DePamphilis, M. L. (1984) J. Mol. Biol. 175, 131-157[CrossRef][Medline] [Order article via Infotrieve]
24. Hay, R. T., and DePamphilis, M. L. (1982) Cell 28, 767-779[CrossRef][Medline] [Order article via Infotrieve]
25. Li, J. J., and Kelly, T. J. (1985) Mol. Cell. Biol. 5, 1238-1246[Abstract/Free Full Text]
26. Roberts, J. D., and Kunkel, T. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7064-7068[Abstract/Free Full Text]
27. Vassilev, L., and Johnson, E. M. (1988) Nucleic Acids Res. 16, 7742[Free Full Text]
28. Pearson, C. E., Wang, Y. H., Griffith, J. D., and Sinden, R. R. (1998) Nucleic Acids Res. 26, 816-823[Abstract/Free Full Text]
29. Pearson, C. E., Eichler, E. E., Lorenzetti, D., Kramer, S. F., Zoghbi, H. Y., Nelson, D. L., and Sinden, R. R. (1998) Biochemistry 37, 2701-2708[CrossRef][Medline] [Order article via Infotrieve]
30. Waga, S., and Stillman, B. (1994) Nature 369, 207-212[CrossRef][Medline] [Order article via Infotrieve]
31. Thomas, D. C., Veaute, X., Kunkel, T. A., and Fuchs, R. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7752-7756[Abstract/Free Full Text]
32. Cordonnier, A. M., and Fuchs, R. P. (1999) Mutat. Res. 435, 111-119[Medline] [Order article via Infotrieve]
33. Pearson, C. E., Ewel, A., Acharya, S., Fishel, R. A., and Sinden, R. R. (1997) Hum. Mol. Genet. 6, 1117-1123[Abstract/Free Full Text]
34. Gordenin, D. A., Kunkel, T. A., and Resnick, M. A. (1997) Nat. Genet. 16, 116-118[CrossRef][Medline] [Order article via Infotrieve]
35. Moore, H., Greenwell, P. W., Liu, C. P., Arnheim, N., and Petes, T. D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1504-1509[Abstract/Free Full Text]
36. Nag, D. K., White, M. A., and Petes, T. D. (1989) Nature 340, 318-320[CrossRef][Medline] [Order article via Infotrieve]
37. Bill, C. A., Taghian, D. G., Duran, W. A., and Nickoloff, J. A. (2001) Mutat. Res. 485, 255-265[Medline] [Order article via Infotrieve]
38. van Den Broek, W. J., Nelen, M. R., Wansink, D. G., Coerwinkel, M. M., te Riele, H., Groenen, P. J., and Wieringa, B. (2002) Hum. Mol. Genet. 11, 191-198[Abstract/Free Full Text]
39. Glazer, P. M., Sarkar, S. N., Chisholm, G. E., and Summers, W. C. (1987) Mol. Cell. Biol. 7, 218-224[Abstract/Free Full Text]
40. Lindahl, T., and Wood, R. D. (1999) Science 286, 1897-1905[Abstract/Free Full Text]
41. North, P., Ganesh, A., and Thacker, J. (1990) Nucleic Acids Res. 18, 6205-6210[Abstract/Free Full Text]
42. Lopez, B., Rousset, S., and Coppey, J. (1987) Nucleic Acids Res. 15, 5643-5655[Abstract/Free Full Text]
43. Datta, H. J., Chan, P. P., Vasquez, K. M., Gupta, R. C., and Glazer, P. M. (2001) J. Biol. Chem. 276, 18018-18023[Abstract/Free Full Text]

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