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Human Molecular Genetics Pages 1117-1123

Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases
Introduction
Results
   Slipped structures
   MSH2 binding
   Binding specificity
Discussion
Materials And Methods
   Plasmids and DNA treatments
   hMSH2 expression and purification
   Band-shift assay
Acknowledgements
References

Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases

Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases Christopher E. Pearson1,*, Amy Ewel2, Samir Acharya2, Richard A. Fishel2,* and Richard R. Sinden1

1Center for Genome Research, Institute of Biosciences and Technology in the Texas Medical Center, Texas A&M University, 2121 West Holcombe, Houston, TX, 77030, USA and 2DNA Repair and Molecular Carcinogenesis Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA

Received February 14, 1997; Revised and Accepted April 9, 1997

The expansion of trinucleotide repeat sequences is associated with several neurodegenerative diseases. The mechanism of this expansion is unknown but may involve slipped-strand structures where adjacent rather than perfect complementary sequences of a trinucleotide repeat become paired. Here, we have studied the interaction of the human mismatch repair protein MSH2 with slipped-strand structures formed from a triplet repeat sequence in order to address the possible role of MSH2 in trinucleotide expansion. Genomic clones of the myotonic dystrophy locus containing disease-relevant lengths of (CTG)n[middot](CAG)n triplet repeats were examined. We have constructed two types of slipped-strand structures by annealing complementary strands of DNA containing: (i) equal numbers of trinucleotide repeats (homoduplex slipped structures or S-DNA) or (ii) different numbers of repeats (heteroduplex slipped intermediates or SI-DNA). SI-DNAs having an excess of either CTG or CAG repeats were structurally distinct and could be separated electrophoretically and studied individually. Using a band-shift assay, the MSH2 was shown to bind to both S-DNA and SI-DNA in a structure-specific manner. The affinity of MSH2 increased with the length of the repeat sequence. Furthermore, MSH2 bound preferentially to looped-out CAG repeat sequences, implicating a strand asymmetry in MSH2 recognition. Our results are consistent with the idea that MSH2 may participate in trinucleotide repeat expansion via its role in repair and/or recombination.

INTRODUCTION

The etiology of nine human genetic diseases, including Huntington's disease, spinocerebellar ataxia type 1 (SCA-1), spino-bulbar muscular atrophy, myotonic dystrophy (DM) and fragile X-syndrome (FRAXA) have been traced to genetic variation in the lengths of (CTG)n[middot](CAG)n or (CGG)n[middot](CCG)n trinucleotide repeats within the affected locus (for a review see ref. 1 ). In normal individuals, these genes contain 5-37 (CTG)n[middot](CAG)n repeats, while increases in the number of repeats to 50-200 are associated with unstable alleles and disease symptoms (1 ,2 ). Explosive expansion to >3000 repeats can occur at the DM and FRAXA loci. The probability of expansion strongly depends upon the number of existing trinucleotide repeats; the longer the repeat tract, the greater the possibility for expansion. This length-sensitive probability of mutation has led to the term `dynamic mutation' to describe this process (3 ). In SCA-1 and FRAXA, the repeat tracts are normally interrupted with the non-repeat units CAT and AGG respectively (4 -7 ). In each case, the non-repeat interruptions confer increased genetic stability to the repeat tract, such that interrupted tracts are less likely to undergo expansions. Loss of non-repeat interruptions, and hence an enhanced length of the pure repeat tracts, are correlated with instability and disease. In the case of FRAXA, changes in the repeat length are polar and occur primarily in the 3' end of the (CGG)n tract, implicating replication direction in the process (6 ). For some of the diseases, there also appear to be specific tissues as well as `windows' for triplet expansion which are believed to occur during gametogenesis and/or early post-zygotic development (1 ).

The mechanism(s) of repeat expansion is unknown. Since both the length and the sequence of the genetically unstable tandem repeats are limited to only certain triplet repeats [(CTG)n[middot](CAG)n, (CGG)n[middot](CCG)n, (AAG)n[middot](CTT)n], it is thought that some type of DNA secondary structure may contribute to trinucleotide instability in these neurodegenerative diseases (1 -13 ). Spontaneous mutations in prokaryotic and eukaryotic (including human) cells frequently are associated with simple DNA repeat sequences that can form alternative non-B DNA structures (reviewed in 10 and 11 ). The formation of slipped-strand structures between repeated trinucleotide sequences is one possible mechanism that may contribute to expansion (Fig. 1 ). Such slipped-strand structures may be formed during DNA replication, and may result in both expansions and deletions of the trinucleotide repeated sequences (Fig. 1 A and B) (12 ,13 ). In addition, slipped-strand structures may also be formed between complementary strands with identical numbers of repeats (Fig. 1 C) (12 ), as may occur intrachromasomally or during meiotic chromosome synapsis. All of these slipped-strand structures contain unpaired nucleotides and may be substrates for mismatch repair (12 ,13 ). Using genomic clones of the DM, SCA-1 and FRAXA loci having normal, pre-mutant and full mutant lengths of repeats, we previously have demonstrated the formation of an apparent slipped-strand structure (12 ,14 , C.E. Pearson unpublished results). In each case, the probability of slipped-strand structure formation increased with increased repeat length. In the case of SCA-1 (CAG)n repeats or FRAXA (CGG)n repeats the presence of CAT or AGG interruptions, respectively, inhibited structure formation (14 , C.E. Pearson unpublished results).These results support the participation of a slipped-strand structure in the process of trinucleotide repeat instability.


Figure 1.Replication slippage models.(A) Slippage of the DNA polymerase and the nascent strand backwards on the template strand would result in the formation of structures containing an excess of repeats on the nascent strand, resulting in expansion products. (B) Slippage of the DNA polymerase and the nascent strand forward on the template strand would result in the formation of structures containing an excess of repeats on the template strand, giving rise to deletion products. Both expansion and deletion processes will give rise to replicated duplex DNAs having a stretch of mismatched repeats (a heteroduplex region). The expected products (in the absence of repair) of two rounds of replication are shown. For simplicity, we have assumed that structure formation occurs only in the lagging strand, although it may occur in either strand. (C) Alternative homoduplex repeat structures may form in non-replicating DNA (12). Thin lines represent repeat tracts, broad lines are non-repetitive flanks.

The human homolog of the Escherichia coli MutS protein, MSH2, is a member of the highly conserved family of mismatch repair proteins (15 ,16 ). In E.coli, MutS initiates mismatch repair via recognition and binding to DNA at the site of a mispair (17 -20 ). The yeast (21 ) and human (22 ,23 ) MSH2 protein also bind specifically to duplex oligonucleotides containing single base pair mismatched nucleotides as well as short (5-16 nucleotide) insertions/deletions loops (IDLs), suggesting that these proteins play a role similar to that of the bacterial MutS. Human tumor cell lines that are deficient in MSH2 display a generalized increase in spontaneous mutation rates, instability in simple repeat (microsatellite) sequences, and yield extracts that are defective for single base pair and IDL mismatch repair in vitro (24 -26 ). Mutations in MSH2 appear to account for ~50% of hereditary nonpolyposis colon cancers (HNPCCs), suggesting that MSH2 plays a central role in carcinogenesis (27 ). Furthermore, hMSH2 forms specific mispair binding complexes with two other MutS homologs, MSH3 (28 ) and MSH6 (GTBP/p160) (28 ,29 ). Although the contribution of mismatch repair to trinucleotide repeat stability in human cells remains controversial (30 ), we have demonstrated that mutations in the bacterial mutS, mutL or mutH genes decrease the frequency of large deletions of plasmid-borne trinucleotide repeats in E.coli (9 ), implicating a functional mismatch repair system in the mechanism of large deletions in bacteria. To address further a possible role for human mismatch repair in trinucleotide repeat expansion, we have investigated the binding of MSH2 to the slipped-strand structures expected to be intermediates in trinucleotide expansions as a model for the fundamental recognition process that is required in mismatch repair.

RESULTS

Slipped structures

Two types of trinucleotide DNA structures were used-homoduplex slipped strand DNA structures (S-DNA) (12 ), formed between two complementary strands having equal numbers of repeats, and heteroduplex slipped intermediate DNA structures (SI-DNA), in which the strands have different numbers of repeats. Two genomic clones derived from the human DM locus that have identical non-repetitive flanking sequences and 30 or 50 triplet repeats (Fig. 2 A) (12 ), corresponding to the normal and expanded alleles (1 ), respectively, were used. Plasmids were linearized by HindIII digestion, 32P labeled (*) on the 3' ends to identical specific activity then treated by denaturation-renaturation (12 ). For the production of S-DNA, only individual plasmids werereduplexed and for SI-DNA equimolar amounts of the 30 and 50 triplet repeat-containing plasmids were mixed and heteroduplexed. Following renaturation, DNAs were digested with EcoRI, resulting in DM inserts uniquely labeled on the 3' end of the CTG strand (12 ). Reduplexing resulted in a reproducible pattern of slow migrating products not present in the non-treated samples (12 ). The ethidium-stained patterns of the reduplexed n = 30 or n = 50 S-DNAs were identical to their respective autoradiographic patterns (Fig. 2 , compare B with C). In the case of reduplexed (CTG)30[middot](CAG)30-containing plasmid, 39% of the repeat-containing fragment migrated as a series of closely spaced distinct products between 232 and 410 bp, with a single region of greatest intensity migrating as 287 bp (Fig. 2 B). For reduplexed (CTG)50[middot](CAG)50-containing plasmid, 70% of the repeat-containing fragment migrated as a series of closely spaced distinct products between 295 and 696 bp, with a single region of greatest intensity migrating as 370 bp (Fig. 2 B). A larger percentage of the DNA containing expanded repeat length (n = 50) formed S-DNA structures than DNA containing repeat lengths found in normal individuals (n = 30) (Fig. 2 B and C, compare lanes 2 and 5), confirming a previously observed relationship that increasing length of trinucleotide repeats results in increased slipped-strand DNA products (12 ).


Figure 2.Reduplexing/heteroduplexing DM DNA fragments results in slowly migrating DNAs. (A) Map of the HindIII-EcoRI (CTG)n[middot](CAG)n-containing fragments from DM genomic clones, where n = 30 and n = 50 (12). Human non-repetitive flanking sequences (positions 357-375 and 391-433, as in ref. 2) are in bold, while the thin lines represent plasmid vector sequences. Positions of the unique 3' CTG radiolabels are indicated (*). LNT, linear non-treated DNAs of n = 30 and n = 50 plasmids (lanes 1 and 4, respectively), reduplexed DNAs (S) from n = 30 and n = 50 plasmids (lanes 2 and 5, respectively), heteroduplexed DNAs (SI) of n = 30 and n = 50 plasmids (lanes 3 and 6, respectively) and the *HindIII-EcoRI pUC19 vector band are indicated. Products were separated on a 4% polyacrylamide gel, stained with ethidium bromide (B) or dried and exposed for autoradiography (C and D). For (B) and (C), samples and lane numbers are identical, and the upper and lower arrows indicate the *(CTG)30[middot](CAG)50 and *(CTG)50[middot](CAG)30 SI-DNAs, respectively. Samples in (D) are gel-purified products from their corresponding lanes in (C). These gel-purified products were loaded in equimolar amounts. Positions of the 123 bp ladder are shown. As previously observed (12), a small amount of conversion to the linear form is evident in both gel-purified S-DNA and SI-DNA samples. The three filled arrowheads indicate post-elution multimeric associations of SI-DNAs, which are more evident with longer exposures.

The ethidium-stained products of the *30*50 or *50*30 heteroduplexing reactions revealed that both n = 30 and n = 50 linear and S-DNA products were formed. The two major slower migrating products and the fainter closely spaced products, that only formed in the reactions that contained both the n = 30 and n = 50 parent DNAs, are the heteroduplex SI-DNAs (Fig. 2 B, see arrows). The ethidium-stained pattern was identical, regardless of whether n = 30 or the n = 50 was radiolabeled (Fig. 2 B). By selectively labeling the (CTG)30- or (CTG)50-containing strands, it is apparent that the uppermost major band represents the *(CTG)30[middot](CAG)50 heteroduplex (Fig. 2 C, lane 3, see arrow), while the faster migrating major band represents the *(CTG)50[middot](CAG)30 heteroduplex (Fig. 2 C, lane 6, see arrow). As with the S-DNA structures formed from reannealing single parent DNAs, there appear to be several structural isomers, with the major product presumably representing the most energetically favorable conformation. Apparently, the difference in the structures of the SI-DNAs containing looped-out CTG or CAG strands are sufficient to confer different electrophoretic mobilities to the sister heteroduplexes, with limited overlap. We previously have demonstrated the electrophoretic resolution of two sister three-way junctions of identical sequence composition (31 ,32 ). For both reduplexing and heteroduplexing reactions, the pattern and relative proportion of products was highly reproducible.

Each of the linear non-treated DNAs, the major S-DNAs and the major SI-DNAs were gel purified (12 ). We found that the gel-purified products were stable as judged by a minimal interconversion back to linear or other forms (compare Fig. 2 C with D) as previously observed (12 ). However, longer autoradiographic exposures (data not shown) revealed that both the isolated SI-DNAs demonstrated some spontaneous self-association to very slow migrating products (positions indicated by arrowheads in Fig. 2 D), whose intensity varied from one preparation to another. It is likely that these represent intermolecular multimeric associations similar to those observed for (CA)n[middot](TG)n and (GA)n[middot](TC)n tracts (33 ,34 ).

MSH2 binding

A band-shift assay was used to detect MSH2 binding to each of the gel-purified substrate DNAs similar to that of Fishel et al. (1994) (22 ,23 ). Under these conditions, MSH2 did not bind significantly to the linear *(CTG)30[middot](CAG)30 or *(CTG)50[middot] (CAG)50 fragments, although, some low level binding was observed (Fig. 3 A, lanes 1-3 and 10-12, see open arrowheads). Binding of hMSH2 to either of the S-DNAs resulted in a single and distinct shifted complex (Fig. 3 A, lanes 4-6 and 13-15, see arrow). MSH2 also bound to the *(CTG)30[middot](CAG)50 SI-DNA, resulting in a similarly shifted complex (Fig. 3 A, lanes 7-9, see arrow). MSH2 bound poorly to the *(CTG)50[middot](CAG)30 SI-DNA (Fig. 3 A, lanes 16-18; Fig. 3 B). These results suggest that purified hMSH2 binds to the slipped-strand S-DNAs and SI-DNAs. Moreover, MSH2 appears to bind the SI-DNAs in a structure- or sequence-specific manner in which there is preferential binding to looped-out CAG repeats (Fig. 3 B and C).


Figure 3. Binding of MSH2 protein to trinucleotide repeat structures. (A) Band-shift assays were performed as described in Materials and Methods. Gel-purified 32P-labeled trinucleotide repeat DNAs in the linear non-treated (LNT), major S-DNA (S) and major SI-DNA (SI) forms, as indicated. The arrows indicate the band-shifted slipped DNA-hMSH2 complexes, while open arrowheads indicate the linear DNA-MSH2 complexes. (B) Binding titration of MSH2 to *(CTG)30[middot](CAG)50 SI-DNA or *(CTG)50[middot](CAG)30 SI-DNA, as indicated, using 0, 485 ng (4.6 pmol), 727.5 ng (6.7 pmol) and 970 ng (9 pmol) of MSH2. (C) Quantification of 3-5 binding assays as shown in (A) and (B), error bars represent plus and minus one standard deviation.

Binding specificity

To test the specificity of MSH2 binding, competition assays were performed (22 ,23 ,31 ). In these studies, the amount of MSH2 bound to *(CTG)30[middot](CAG)30 S-DNA, *(CTG)50[middot](CAG)50 S-DNA, or *(CTG)30[middot](CAG)50 SI-DNA was examined, when excess gel-purified linear non-treated DNAs, the S-DNAs or the SI-DNAs was introduced into the binding reaction. Such a competition reaction, where MSH2 binding to the *(CTG)30[middot](CAG)50 SI-DNA is examined, is shown in Figure 4 A and B. We found that the linear (CTG)30[middot](CAG)30 DNA was a poor competitor, while the S-DNA and both SI-DNAs were effective competitors of MSH2 binding. At low concentrations (50-fold molar excess), the (CTG)30[middot](CAG)50 SI-DNA was more effective than the (CTG)50[middot](CAG)30 SI-DNA as competitor (Fig. 4 B-D). The efficiency of each competitor for MSH2 binding to *(CTG)30[middot](CAG)30 S-DNA (Fig. 4 C) or *(CTG)50[middot](CAG)50 S-DNA (Fig. 4 D) was similar to that observed for binding to *(CTG)30[middot](CAG)50 SI-DNA (Fig. 4 B). These results suggest that the binding of MSH2 to S-DNA and SI-DNAs is specific and is similar to the binding of hMSH2 to single base pair and IDL mismatched nucleotides.


Figure 4. Structure-specific binding by human MSH2 protein. DNA competition assays were performed (23, 24) using gel-purified linear non-treated DNAs (LNT), S-DNAs (S) and SI-DNAs (SI) as competitors. Binding was done as in Figure 3 using 0 and 727.5 ng (6.7 pmol) of MSH2. After binding, the quantity of competitor indicated was added and the mixture was incubated for an additional 15 min. Reaction products were separated by gel electrophoresis, the gels were dried and autoradiographed. (A) Autoradiograph of *(CTG)30[middot](CAG)50 SI-DNA-MSH2 complex competition assay with 100 and 200 molar-fold amount of competitor; no protein (NP), no competitor (NC). The arrow indicates the band-shifted MSH2-DNA complex. (B) Quantification of *(CTG)30[middot](CAG)50 SI-DNA-MSH2 complex competition assay, with 50, 100 and 200 molar-fold amount of competitor. (C) Quantification of *(CTG)30[middot](CAG)30 S-DNA-hMSH2 complex competition assay. (D) Quantification of *(CTG)50[middot](CAG)50 S-DNA-MSH2 complex competition assay. Quantification of 3-5 competition assays as shown; error bars represent plus and minus one standard deviation.

To address further the possibility that there is an intrinsic difference in the ability of MSH2 to recognize CTG and CAG intrastranded structures, we tested MSH2 binding to synthetic (CTG)15 and (CAG)15 oligonucleotides (Fig. 5 ). We found that MSH2 bound to the (CAG)15 oligonucleotide 4- to 5-fold more efficiently than to the (CTG)15 oligonucleotide. This result is similar to the observed preferential binding of the *(CTG)30[middot](CAG)50 SI-DNA (Fig. 3 B), which has an excess of 20 CAG repeats, and suggests that there is an intrinsic difference in the recognition of CAG trinucleotide repeat sequences over CTG trinucleotide repeat sequences. These results also suggest that on either of the homoduplex S-DNAs it is the looped-out CAG, and not the looped-out CTG strand that is being bound preferentially by MSH2.


Figure 5.Human MSH2 protein binding to (CAG)15 and (CTG)15. (A) Individual oligonucleotides were twice purified by TLC (SurePure, USB) and radiolabeled to identical specific activity. Binding assays were performed as in Figure 3. DNAs were incubated with 0, 485 ng (4.6 pmol) and 727.5 ng (6.7 pmol) of MSH2. Products were separated on a 4% polyacrylamide gel, dried and exposed for autoradiography. (B) Quantification of 3-5 binding assays as shown in (A); error bars represent plus and minus one standard deviation.

DISCUSSION

The yeast and human MSH2 proteins are required for the recognition of single base pair mismatched nucleotides, duplex oligonucleotides containing short IDLs, palindromic DNA IDL structures and Holliday junctions (21 -23 ,35 ). Both purified proteins demonstrated a binding preference for the IDL substrates, and binding appeared to increase with increased loop size. Here, we observed significant and specific MSH2 binding to both slipped-strand S-DNAs and SI-DNAs and minimal binding to linear duplex DNAs. There appears to be a binding hierarchy with (CTG)50[middot] (CAG)50 S-DNA>(CTG)30[middot](CAG)30 S-DNA ~(CTG)30[middot](CAG)50 SI-DNA>(CTG)50[middot](CAG)30 SI-DNA>>(CTG)50[middot](CAG)50 linear = (CTG)30[middot](CAG)30 linear. In addition, we found a direct association between MSH2 binding and the number of trinucleotide repeats. This relationship parallels the effect of repeat length on instability in vivo. The KD of hMSH2 binding to these trinucleotide repeat structures was calculated to be in the high nanomolar range (50-200 nM), which is similar to the mispair binding affinity exhibited by the bacterial MutS protein and our previously reported MSH2 mispair binding affinity (36 ).

The structure(s) within the S-DNA and SI-DNA substrates that are recognized by MSH2 are unknown. However, these structures are likely to be somewhat different from the IDL substrates that previously were shown to be recognized efficiently by the human MSH2 protein (22 ,23 ). This is because the slipped-strand DNA structures formed within both S-DNA and SI-DNA substrates are capable of branch migration, which can contribute to the production of a wide variety of structural isomers. Additionally, the looped-out region(s) formed by SI-DNAs are significantly larger than that used in our previous experiments (21 -23 ). Several studies have shown that the efficiency and specificity of substrate recognition and correction varies with the mismatch, loop, DNA structure (hairpin, palindrome or chemical modification) and the sequence context surrounding the mismatch (17 -23 ,26 ,35 -38 ). These results suggest that MSH2 may recognize a variety of mispair and DNA structures with wide-ranging affinity. It is interesting to note that all of the slipped-strand structures tested here gave rise to a single similarly shifted complex. There are a number of possible DNA structures that might be recognized by MSH2 within these slipped-strand substrates and include: (i) the junction of the interstrand region with the looped-out strand; (ii) a CAG or CTG hairpin/loop; (iii) an A[middot]A, T[middot]T, C[middot]C, C[middot]T, A[middot]C, G[middot]G A[middot]G or G[middot]T mismatch(es) contained in perfectly or imperfectly annealed CTG or CAG hairpins; or (iv) the tip of an intrastrand hairpin. The binding to the (CAG)15 and (CTG)15 oligos appears to diminish a loop junction or three-way junction as the only recognition site since these oligonucleotides are not large enough to form such structures.

Our studies show a clear MSH2 binding preference for the CAG strand. Presumably some, as yet undefined, difference in the structure of the two looped-out CTG and CAG strands accounts for the difference in the recognition by MSH2. While both CTG and CAG DNA strands will form hairpins, the stability of the CTG hairpin is greater than that of the CAG hairpin (39 -41 ). We have shown previously that the CAG strand in the S-DNAs is preferentially sensitive to the single-strand-specific mung bean nuclease (12 ), suggesting a greater single-stranded character of the looped-out/hairpin CAG relative to the CTG strand. Perfect hairpins formed by the CAG and CTG strands would contain an A[middot]A and T[middot]T mispair respectively, flanked by two C[middot]G base pairs. In simian cells, A[middot]A and T[middot]T mismatches are corrected with 64 and 39% efficiency, respectively (37 ). Thus, if only these mispairs in their respective looped-out stems were recognized, the preferential binding of MSH2 to the slipped-strand intermediate with an excess of CAG correlates with the efficiency of mispair correction in vivo.

Several studies have shown that trinucleotide repeat sequences are unstable to a degree greater than that observed for mono- and dinucleotide repeat tracts in tumors or tumor cell lines that display microsatellite instability (42 ,43 ). Furthermore, the relative frequency of insertions versus deletions differed depending on the sequence of the repeat tract (43 ). Interestingly, neither the DM nor the FRAXA repeat loci displayed large repeat expansions in the MSH2-deficient human tumor cell line Lovo, although shorter instabilities of these repeats was observed (30 ). These results suggested that an MSH2 deficiency alone does not cause the large changes in trinucleotide repeat lengths typical of DM and FRAXA individuals. However, it is possible for the post-replication mismatch repair process to be unrelated to large trinucleotide expansions, while the mismatch repair genes (proteins) may play a fundamental role. For example, the mismatch repair genes have been found to participate in several other DNA metabolic processes, such as transcription-coupled repair (TCR) (44 ,45 ) and the elimination of genetic recombination between the divergent DNA sequences (termed: homoeologous recombination) (46 ,47 ). These latter studies have suggested that the mismatch repair proteins may be part of a system that alters or aborts recombination events initiated between divergent repeated sequences, thereby maintaining the genetic distinction of these sequences as well as the global integrity of chromosome containing such repeats. Furthermore, genetic studies suggest that Saccharomyces cerevisiae MSH2-dependent mismatch repair controls the length of a mitotic genetic recombination tract (48 ,49 ). Thus, the observed lack of large trinucleotide expansion in mismatch repair-deficient human tumor cell lines may not address directly the possibility of tissue specificity, altered expression and or mutation of MSH2 during development, the contribution(s) of transcription/TCR, or cells undergoing high rates of recombination as a causes of trinucleotide expansion. Furthermore, while HNPCC patients do not appear to be predisposed to the neurodegenerative diseases associated with expansion of trinucleotide repeat sequences, it should be noted that they are heterozygous carriers, while only homozygous or hemizygous mutation(s) of the mismatch repair genes result in a mismatch repair deficiency.

Mismatch repair in human cells appears to be far more complicated than in bacteria. It is now clear that MSH2 forms at least two heteromeric protein complexes that enhance the specificity of MSH2-dependent mispair recognition (28 ,29 ,50 ). These two complexes have overlapping and redundant mispair binding activities (28 ) that appear to account for the lack of observed mutations in MSH6 and, by analogy MSH3, in HNPCC. Furthermore, while the binding of MSH2 alone appears to be somewhat lower than MSH2 complexed with either MSH3 or MSH6, its fundamental mispair binding activity appears to completely recapitulate the types of binding observed when it is complexed with either MSH3 or MSH6. The enhancement of mispair binding observed for the MSH2-MSH6 and MSH2-MSH3 protein complexes over MSH2 alone has suggested that MSH3 and MSH6 are `specificity factors' that increase the stability of the MSH2 mispair binding activity (28 ). These observations suggest that MSH2 may provide a model system for mispair binding in the absence of clear knowledge of the appropriate protein specificity partner and that the association of MSH3 and/or MSH6 with MSH2 may broaden the number of mismatch repair proteins (genes) that might contribute to trinucleotide repeat expansion.

Our results suggest that slipped intermediates having an excess of CTG repeats may escape detection more frequently than those with CAG repeats. This asymmetry may indicate that it is the CTG hairpin, rather than the CAG hairpin, which determines trinucleotide instability at neurodegenerative disease loci. These results appear to parallel the observation that certain palindromic sequences can escape repair in yeast and mammalian cells (51 ,52 ). An alternative possibility is that MSH2 either alone or in a complex form may stabilize or protect slipped-strand CAG loops from repair, thus allowing replication to finalize an expansion. It is also possible that MSH2 binding to the slipped-strand CAG loops exposes the looped-out CTG strand to resolvases. Such asymmetric recognition may account for strand-specific differences in the replication fidelity (8 ,53 ), inequality in mutation rates between complementary DNA strands (54 ,55 ) and perhaps the polar mutability of trinucleotide repeat tracts observed in vivo (5 -8 ).

Finally, our observation that MSH2 binding increases with increasing length of the slipped-strand structure suggests that the role of MSH2 in trinucleotide expansion may become more important with longer repeat tracts. Whether MSH2 or other mismatch repair factors are intimately involved in the biology of trinucleotide expansion clearly requires further study.

MATERIALS AND METHODS

Plasmids and DNA treatments

The DM clones have been described previously (12 ) (see also Fig. 2 ). Isolation, radiolabeling and gel purification were as described (12 ,31 ,32 ). Denaturation and renaturation for reduplexing and heteroduplexing DNAs was by alkali treatment (pH 13) and neutralization (pH 8) with T.E. at 68oC, as described in detail (12 ).

hMSH2 expression and purification

MSH2 protein was expressed in a baculovirus system and purified by a modification of the procedure described by Fishel et al. (28 ).

Band-shift assay

Band-shift assays were performed (23 ,24 ) using 1.9 pmol/reaction and 2.4 pmol/reaction of gel-purified 32P-labeled 30*30 or 50*50 DNAs, or 1.4 pmol/reaction of oligonucleotide. DNAs were incubated with 0-970 ng (0-9 pmol) of purified MSH2 (97 ng/[mu]l) in binding buffer [20 mM potassium phosphate (pH 7.5), 50 mM KCl, 0.1 mM dithiothreitol (DTT), 1 mM EDTA, and 23% glycerol], plus 150 pmol of double-stranded poly(dI-dC) (Pharmacia) in 20 [mu]l reactions, and incubated at 25oC for 15 min. Reaction products were separated on a 4% polyacrylamide gel, dried and exposed for autoradiography as described (23 ,24 ). All quantification was by PhosphorImage analysis using ImageQuant software (Molecular Dynamics) and represent 3-5 experiments.

ACKNOWLEDGEMENTS

We thank Tetsuo Ashizawa, Fred Gimble, David L. Nelson, Vladimir Potaman, Stephen T. Warren and Samuel Wilson for critical comments on the manuscript. This work was funded in part by funds from the Institute of Biosciences and Technology and from NIH grant GM52982 (R.R.S.) and CA67007 (R.F.).

REFERENCES

1 Ashley, C.T.,Jr and Warren, S.T. (1995) Trinucleotide repeat expansion and human disease. Annu. Rev. Genet., 29, 703-728. MEDLINE Abstract

2 Mahadevan, M., Tsilfidis, C., Sabourin, L., Shutler, G., Amemiya, C., Jansen, G., Neville, C., Narang, M., Barceló, J., O'Hoy, K., LeBlond, S., Earle-MacDonald, J., DeJong, P.J., Wieringa, B. and Korneluk, R.G. (1992) Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. Science, 255, 1253-1255. MEDLINE Abstract

3 Richards, R.I. and Sutherland, G.R. (1992) Heritable unstable DNA sequences. Nature Genet., 1, 7-9. MEDLINE Abstract

4 Chung, M.-y., Ranum, L.P.W., Duvick, L.A., Servadio, A., Zoghbi, H.Y. and Orr, H.T. (1993) Evidence for a mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type I. Nature Genet., 5, 254-258.

5 Eichler, E.E., Holden, J.J.A., Popovich, B.W., Reiss, A.L., Snow, K., Thibodeau, S.N., Richards, C.S., Ward, P.A. and Nelson, D.L. (1994) Length of uninterrupted CGG repeats determines instability in the FMR1 gene. Nature Genet., 8, 88-94. MEDLINE Abstract

6 Kunst, C.B. and Warren, S.T. (1994) Cryptic and polar variation in the fragile X repeat could result in predisposing normal alleles. Cell, 77, 853-861. MEDLINE Abstract

7 Snow, K., Tester, D.J., Kruckeberg, K.E., Schaid, D.J. and Thibodeau, S.N. (1994) Sequence analysis of the fragile X trinucleotide repeat: implications for the origin of the fragile X mutation. Hum. Mol. Genet., 9, 1543-1551.

8 Kang, S., Jaworski, A., Oshima, K. and Wells, R.D. (1995) Expansion and deletion of CTG repeats from human disease genes are determined by the direction of replication in E.coli. Nature Genet., 10, 213-218. MEDLINE Abstract

9 Jaworski, A., Rosche, W.A., Gallibolian, R., Kang, S., Shimizu, M., Bowater, R.P., Sinden, R.R. and Wells, R.D. (1995) Mismatch repair in Escherichia coli enhances instability of (CTG)n triplet repeats from human hereditary diseases. Proc. Natl Acad. Sci. USA, 92, 11019-11023. MEDLINE Abstract

10 Sinden, R.R. and Wells, R.D. (1992) DNA structure, mutations, and human genetic disease. Curr. Opin. Biotechnol., 3, 612-622. MEDLINE Abstract

11 Wells, R.D. and Sinden, R.R. (1993) In Davies, K.E. and Warren, S.T. (eds), Genome Analysis, Genome Rearrangement and Stability. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, vol. 7, pp. 107-138.

12 Pearson, C.E. and Sinden, R.R. (1996) Alternative structures in duplex DNA formed within the trinucleotide repeats of the myotonic dystrophy and fragile X loci. Biochemistry, 35, 5041-5053. MEDLINE Abstract

13 Richards, R.I. and Sutherland, G.R. (1994) Simple repeat DNA is not replicated simply. Nature Genet., 6, 114-116. MEDLINE Abstract

14 Pearson, C.E. Eichler, E.E., Lorenzetti, D., Acharya, S., Kramer, P.R., Kramer, S.F., Nelson, D.L., Zoghbi, H.Y. and Sinden, R.R. (1996) Slipped strand structures in neurodegenerative disease-associated trinucleotide repeats: a role for human mismatch repair and cryptic interruptions. Am. J. Hum. Genet., 59 (suppl.), A48, 244.

15 Fishel, R., Lescoe, M.K., Rao, M.R.S., Copeland, N.G., Jenkins, N.A., Garber, J., Kane, M. and Kolodner, R. (1993) The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell, 75, 1027-1038. MEDLINE Abstract

16 Leach, F.S. et al., (1993) Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell, 75, 1215-1225. MEDLINE Abstract

17 Su, S.-S. and Modrich, P. (1986) Escherichia coli mutS-encoded protein binds to mismatched DNA basepairs. Proc. Natl Acad. Sci. USA, 83, 5057-5061.

18 Su, S.-S., Lahue, R.S., Au, K.G. and Modrich, P. (1988) Mispair specificity of methyl-directed DNA mismatch correction in vitro. J. Biol. Chem., 263, 6829-6835.

19 Jiricny, J., Su, S.-S., Wood, S.G. and Modrich, P. (1988) Mismatch-containing oligonucleotide duplexes bound by the E.coli mutS-encoded protein. Nucleic Acids Res., 16, 7843-7853. MEDLINE Abstract

20 Parker, B.O. and Marinus, M.G. (1992) Repair of DNA heteroduplexes containing small heterologous sequences in Escherichia coli. Proc. Natl Acad. Sci. USA, 89, 1730-1734. MEDLINE Abstract

21 Alani, E., Chi, N.-W. and Kolodner, R.D. (1995)The Saccharomyces cerevisiae Msh2 protein specifically binds to duplex oligonucleotides containing mismatched DNA base pairs and insertions. Genes Dev., 9, 234-247. MEDLINE Abstract

22 Fishel, R., Ewel, A. and Lescoe, M.K. (1994) Purified human MSH2 protein binds to DNA containing mismatched nucleotides. Cancer Res., 54, 5539-5542. MEDLINE Abstract

23 Fishel, R., Ewel, A., Lee, S., Lescoe, M.K. and Griffith, J. (1994) Binding of mismatched microsatellite DNA sequences by the human MSH2 protein. Science, 266, 1403-1405. MEDLINE Abstract

24 Bhattacharyya, N.P., Skandalis, A., Ganesh, A., Groden, J. and Meuth, M. (1994) Mutator phenotype in human colorectal carcinoma cell lines. Proc. Natl Acad. Sci. USA, 91, 6319-6323. MEDLINE Abstract

25 Parsons, R., Li, G.-M., Longely, M.J., Fang, W.-H., Papadopoulos, N., Jen, J., de la Chapelle, A., Kinzler, K.W., Vogelstein, B. and Modrich, P. (1993) Hypermutability and mismatch repair deficiency in RER+ tumor cells. Cell, 75, 1227-1236 MEDLINE Abstract

26 Umar, A., Boyer, J.C. and Kunkel, T.A. (1994) DNA loop repair by human cell extracts. Science, 266, 814-816. MEDLINE Abstract

27 Fishel, R. and Kolodner, R.D. (1995) Identification of mismatch repair genes and their role in the development of cancer. Curr. Opin. Genet. Dev., 5, 382-395. MEDLINE Abstract

28 Acharya, S., Wilson, T., Gradia, S., Kane, M.F., Guerrette, S., Marsischky, G.T., Kolodner, R. and Fishel, R. (1996) hMSH2 forms specific mispair-binding complexes with hMSH3 and hMSH6. Proc. Natl Acad. Sci. USA, 93, 13629-13634. MEDLINE Abstract

29 Drummond, J.T., Li, G.-M., Longley, M.J. and Modrich, P.(1995) Isolation of an hMSH2-p160 heterodimer that restores DNA mismatch repair to tumor cells. Science, 268, 1909-1912. MEDLINE Abstract

30 Kramer, P.R., Pearson, C.E. and Sinden, R.R. (1996) Stability of triplet repeats of myotonic dystrophy and fragile X loci in human mutator mismatch repair cell lines. Hum. Genet., 98, 151-157. MEDLINE Abstract

31 Pearson, C.E., Ruiz, M.T., Price, G.B. and Zannis-Hadjopoulos, M. (1994) Cruciform DNA binding protein in HeLa cell extracts. Biochemistry, 33, 14185-14196. MEDLINE Abstract

32 Pearson, C.E., Zannis-Hadjopoulos, M., Price, G.B. and Zorbas, H. (1994) A novel type of interaction between a cruciform DNA structure and a cruciform binding protein from HeLa cells. EMBO J., 14, 1571-1580.

33 Gaillard, C. and Strauss, F. (1994) Association of poly(CA)[middot](poly(TG) DNA fragments into four-stranded complexes bound by HMG1 and 2. Science, 264, 433-436. MEDLINE Abstract

34 Belotserkovskii, B.P. and Johnston, B.H. (1996) Polypropylene tube surfaces may induce denaturation and multimerization of DNA. Science, 271, 222-223. MEDLINE Abstract

35 Alani, E., Lee, S., Kane, M.F., Griffith, J. and Kolodner, R. (1997) Saccharomyces cerevisiae MSH2, a mispair recognition protein, also recognizes Holliday junctions. J. Mol. Biol., 265, 289-301 MEDLINE Abstract

36 Mello, J.A., Acharya, S. Fishel, R. and Essigman, J.M. (1996) The mismatch repair protein hMSH2 binds selectively to DNA adducts of the anticancer drug cisplatin. Chem. Biol., 3, 579-589. MEDLINE Abstract

37 Brown, T.C. and Jiricny, J. (1988) Different base/base mispairs are corrected with different efficiencies and specificities in monkey kidney cells. Cell, 54, 705-711. MEDLINE Abstract

38 Jones, M., Wagner, R. and Radman, M. (1987) Repair of a mismatch is influenced by the base composition of the surrounding nucleotide sequence. Genetics, 115, 605-610. MEDLINE Abstract

39 Gacy, A.M., Goellner, G., Juranic, N., Macura, S. and McMurray, C.T. (1995) Trinucleotide repeats that expand in human disease form hairpin structures in vitro. Cell, 81, 533-540. MEDLINE Abstract

40 Mitas, M., Yu, A., Dill, J., Kamp, T.J., Chambers, E.J. and Haworth, I.S. (1995) Hairpin properties of single-stranded DNA containing a GC-rich triplet repeat: (CTG)15. Nucleic Acids Res., 23, 1050-1059. MEDLINE Abstract

41 Smith, G.K., Jie, J., Fox, G.E. and Gao, X. (1995) DNA CTG triplet repeats involved in dynamic mutations of neurobiologically related gene sequences form stable duplexes. Nucleic Acids Res., 23, 4303-4311. MEDLINE Abstract

42 Wooster, R., Cleton-Jansen, A.-M., Collins, N., Mangion, J., Cornelis, R.S., Cooper, C.S., Gusterson, B.A., Ponder, B.A.J., von Demling, A., Wiestler, O.D., Cornelisse, C.J., Devilee, P. and Stratton, M.R. (1994) Instability of short tandem repeats (microsatellites) in human cancers. Nature Genet., 6, 152-156. MEDLINE Abstract

43 Shibata, D., Peinado, M.A., Ionov, Y., Malkhosyan, S. and Perucho, M. (1994) Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation. Nature Genet., 6, 273-281. MEDLINE Abstract

44 Mellon, I. and Champe, G.N. (1995) Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide excision repair of the lactose operon in Escherichia coli. Proc. Natl Acad. Sci. USA, 93, 1292-1297.

45 Mellon, I., Deepak, K.R., Koi, M.C., Boland, R.C. and Champe, G.N. (1996) Transcription-coupled repair deficiency and mutations in human mismatch repair genes. Science, 272, 557-560. MEDLINE Abstract

46 Rayssiguier, C., Thaler, D.S. and Radman, M. (1989) The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature, 342, 396-401. MEDLINE Abstract

47 Bailis, A.M. and Rothstein, R. (1990) A defect in mismatch repair in Saccharomyces cerevisiae stimulates ectopic recombination between homeologous genes by an excision repair dependent process. Genetics, 126, 535-547. MEDLINE Abstract

48 Alani, E., Reenan, R.A.G. and Kolodner, R. (1994) Interaction between mismatch repair and genetic recombination in Saccharomyces cerevisiae. Genetics, 137, 19-39. MEDLINE Abstract

49 Datta, A., Adjiri, A., New, L., Crouse, G.F. and Jinks-Robertson, S. (1996) Meiotic crossovers between divergent sequences are regulated by mismatch repair proteins in Saccharomyces cerevisiae. Mol. Cell. Biol., 16, 1085-1093. MEDLINE Abstract

50 Palombo, F., Gallinari, P., Iaccarino, I., Lettieri, T., Hughes, M., D'Arrigo, A., Truong, O., Hsuan, J.J. and Jiricny, J. (1995) GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells. Science, 268, 1912-1914. MEDLINE Abstract

51 Nag, D.K., White, M.A. and Petes, T.D. (1989) Palindromic sequences in heteroduplex DNA inhibit mismatch repair in yeast. Nature, 340, 318-320. MEDLINE Abstract

52 Bollag, R.J., Elwood, D.R., Tobin, E.D., Godwin, A.R. and Liskay, R.M. (1992) Formation of heteroduplex DNA during mammalian intrachromasomal gene conversion. Mol. Cell. Biol., 12, 1546-1552. MEDLINE Abstract

53 Bohr, V.A. (1991) Gene specific DNA repair. Carcinogenesis, 12, 1983-1992. MEDLINE Abstract

54 Trinh, T.Q. and Sinden, R.R. (1991) Preferential DNA secondary structure mutagenesis in the lagging strand of replication in E.coli. Nature, 352, 544-547. MEDLINE Abstract

55 Wu, C.-I. and Maeda, N. (1987) Inequality in mutation rates of the two strands of DNA. Nature, 327, 169-170.

*To whom correspondence should be addressed. C.E.P. Tel: +1 713 677 7676; Fax: +1 713 677 7689; Email: cpearson{at}ibt.tamu.edu, or R.F. Tel: +1 215 503 1345; Fax: +1 215 923 1098; Email: rfishel{at}hendrix.jci.tju.edu

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