Base stacking and even/odd behavior of hairpin loops in DNA triplet repeat slippage and expansion with DNA polymerase

Repetitions of CAG or CTG triplets in DNA can form intrastrand hairpin loops with combinations of normal and mismatched base pairs that easily rearrange. Such loops may promote primer-template slippage in DNA replication or repair to give triplet-repeat expansions like those associated with neurodegenerative diseases. Using self-priming sequences (e.g. (CAG)(16)(CTG)(4)), we resolve all hairpin loops formed and measure their slippage and expansion rates with DNA polymerase at 37 degrees C. Comparing CAG/CTG loop structures with GAC/GTC structures, having similar hydrogen bonding but different base stacking, we find that CAG, CTG, and GTC triplets predominantly form even-membered loops that slip in steps of two triplets, whereas GAC triplets favor odd-numbered loops. Slippage rates decline as hairpin stability increases, supporting the idea that slippage initiates more easily in less stable regions. Loop stabilities (in low salt) increase in the order GTC < CAG < GAC < CTG, while slippage rates decrease in the order GTC > CAG approximately GAC > CTG. Loops of GTC compared with CTG melt 9 degrees C lower and slip 6-fold faster. We interpret results in terms of base stacking, by relating melting temperature to standard enthalpy changes for doublets of base pairs and mispairs, considering enthalpy-entropy compensation.     

The Effects of Trinucleotide Repeats Found in Human Inherited Disorders on Palindrome Inviability in Escherichia Coli Suggest Hairpin Folding Preferences in Vivo

Unusual DNA secondary structures have been implicated in the expansion of trinucleotide repeat tracts that are associated with several human inherited disorders. We present evidence consistent with the folding of these trinucleotide repeats into hairpin loops at the center of a long DNA palindrome in vivo. Our assay utilizes a palindrome in bacteriophage λ, the center of which determines its ability to inhibit plaque formation in a manner that is consistent with folding into a hairpin or cruciform structure. We show that central inserts of even numbers of d(CAG)·d(CTG) repeats inhibit plaque formation more than do odd numbers. Both d(CAG)(2)·d(CTG)(2) and d(CGG)(2)·d(CCG)(2) central sequences behave like DNA sequences known to form two-base loops in vitro, suggesting that they may also form compact and stable loops. By contrast, repeats of d(GAC)·d(GTC) do not show any evidence consistent with unusual loop stability. These results agree with in vitro evidence that the unstable repeats can form hairpin secondary structures and suggest a favored position of folding. We discuss the potential roles of secondary structures, DNA replication and recombination in models of repeat tract expansion.     

79 Thermodynamic and kinetic studies of trinucleotide repeat (TNR) DNA

The expansion of trinucleotide repeat (TNR) DNA has been linked to several neurodegenerative diseases (McMurray, 2010). The number of repeats is usually a characteristic indication of the severity of TNR-related diseases, with longer repeats giving higher propensity to expand and earlier onset of symptoms (López, Cleary, & Pearson, 2010). It is generally accepted that formation of noncanonical secondary structures, such as stem-loop hairpins or slipouts, contributes to the expansion mechanisms during aberrant DNA replication or repair processes (Mirkin, 2007). The stability of these hairpins is considered an important factor (Paiva & Sheardy, 2005). In this work, we used differential scanning calorimetry (DSC) and UV–Vis spectroscopy to study the thermodynamic and kinetic stability of a series of (CTG)_n and (CAG)_n TNR stem-loop hairpins and their corresponding (CTG)_n/(CAG)_n duplexes (n?=?6–14). We found that hairpins with n?=?even and n?=?even?+?1 (odd) repeats possess very similar thermodynamic stability. But, when converting to the canonical duplex form, odd-repeat hairpins are more stabilized compared to those of their even-repeat counterparts. Within both even- and odd-repeat series, hairpins with longer repeats are thermodynamically more stabilized compared to the shorter ones. Kinetic experiments of the stem-loop hairpin to duplex conversion revealed a longer lifetime for the even-repeat hairpins, while the odd-repeat hairpins convert to duplexes 10-fold faster. Also, hairpins with increased number of repeats are more resistant to the conversion when considered within the even- or odd-repeat series individually. Taken together, although it is thermodynamically more favored that hairpins containing longer repeats convert to canonical duplex form; On the contrary, these longer hairpins are kinetically trapped during the conversion and therefore can persist the noncanonical structures, which allows TNR expansion.     

Trinucleotide repeats associated with human disease.

Triplet repeat expansion diseases (TREDs) are characterized by the coincidence of disease manifestation with amplification of d(CAG. CTG), d(CGG.CCG) or d(GAA.TTC) repeats contained within specific genes. Amplification of triplet repeats continues in offspring of affected individuals, which generally results in progressive severity of the disease and/or an earlier age of onset, phenomena clinically referred to as 'anticipation'. Recent biophysical and biochemical studies reveal that five of the six [d(CGG)n, d(CCG)n, (CAG)n, d(CTG)n and d(GAA)n] complementary sequences that are associated with human disease form stable hairpin structures. Although the triplet repeat sequences d(GAC)n and d(GTC)n also form hairpins, repeats of the double-stranded forms of these sequences are conspicuously absent from DNA sequence databases and are not anticipated to be associated with human disease. With the exception of d(GAG)n and d(GTG)n, the remaining triplet repeat sequences are unlikely to form hairpin structures at physiological salt and temperature. The details of hairpin structures containing trinucleotide repeats are summarized and discussed with respect to potential mechanisms of triplet repeat expansion and d(CGG.CCG) n methylation/demethylation.     

Structure of even/odd trinucleotide repeat sequences modulates persistence of non-B conformations and conversion to duplex

Expansion of trinucleotide repeats (TNR) has been implicated in the emergence of neurodegenerative diseases. Formation of non-B conformations such as hairpins by these repeat sequences during DNA replication and/or repair has been proposed as a contributing factor to expansion. In this work we employed a combination of fluorescence, chemical probing, optical melting, and gel shift assays to characterize the structure of a series of (CTG)(n) sequences and the kinetic parameters describing their interaction with a complementary sequence. Our structure-based experiments using chemical probing reveal that sequences containing an even or odd number of CTG repeats adopt stem-loop hairpins that differ from one another by the absence or presence of a stem overhang. Furthermore, we find that this structural difference dictates the rate at which the TNR hairpins convert to duplex with a complementary CAG sequence. Indeed, the rate constant describing conversion to (CAG)(10)/(CTG)(n) duplex is slower for sequences containing an even number of CTG repeats than for sequences containing an odd number of repeats. Thus, when both the CAG and CTG hairpins have an even number of the repeats, they display a longer lifetime relative to when the CTG hairpin has an odd number of repeats. The difference in lifetimes observed for these TNR hairpins has implications toward their persistence during DNA replication or repair events and could influence their predisposition toward expansion. Taken together, these results contribute to our understanding of trinucleotide repeats and the factors that regulate persistence of hairpins in these repetitive sequences and conversion to canonical duplex.     

Coordinated Processing of 3′ Slipped (CAG)n/(CTG)n Hairpins by DNA Polymerases β and δ Preferentially Induces Repeat Expansions

Expansion of CAG/CTG trinucleotide repeats causes certain familial neurological disorders. Hairpin formation in the nascent strand during DNA synthesis is considered a major path for CAG/CTG repeat expansion. However, the underlying mechanism is unclear. We show here that removal or retention of a nascent strand hairpin during DNA synthesis depends on hairpin structures and types of DNA polymerases. Polymerase (pol) δ alone removes the 3′-slipped hairpin using its 3′-5′ proofreading activity when the hairpin contains no immediate 3′ complementary sequences. However, in the presence of pol β, pol δ preferentially facilitates hairpin retention regardless of hairpin structures. In this reaction, pol β incorporates several nucleotides to the hairpin 3′-end, which serves as an effective primer for the continuous DNA synthesis by pol δ, thereby leading to hairpin retention and repeat expansion. These findings strongly suggest that coordinated processing of 3′-slipped (CAG)_n/(CTG)_n hairpins by polymerases δ and β on during DNA synthesis induces CAG/CTG repeat expansions.     

Srs2 helicase of Saccharomyces cerevisiae selectively unwinds triplet repeat DNA

Trinucleotide repeat expansions are the mutational cause of at least 15 genetic diseases. In vitro, single-stranded triplet repeat DNA forms highly stable hairpins, depending on repeat sequence, and a strong correlation exists between hairpin-forming ability and the risk of expansion in vivo. Hairpins are viewed, therefore, as likely mutagenic precursors to expansions. If a helicase unwinds the hairpin, it would be less likely to expand. Previous work indicated that yeast Srs2 DNA helicase selectively blocks expansions in vivo (Bhattacharyya, S., and Lahue, R. S. (2004) Mol. Cell. Biol. 24, 7324-7330). For example, srs2 mutants, including an ATPase-defective point mutant, exhibit substantially higher expansion rates than wild type controls. In contrast, mutation of another helicase gene, SGS1, had little effect on expansion rates. These findings prompted the idea that Srs2 might selectively unwind triplet repeat hairpins. In this study, DNA helicase assays were performed with purified Srs2, Sgs1, and Escherichia coli UvrD (DNA helicase II). Srs2 shows substantially faster unwinding than Sgs1 or UvrD on partial duplex substrates containing (CTG) x (CTG) sequences, provided that Srs2 encounters the triplet repeat DNA immediately on entering the duplex. Srs2 was also faster at unwinding (CAG) x (CAG)- and (CCG) x (CCG)-containing substrates and an intramolecular (CTG) x (CTG) hairpin. In contrast, all three enzymes unwind about equally well control substrates with either Watson-Crick base pairs or mismatched substrates with non-CNG repeats. Overall, the selective unwinding activity of Srs2 on triplet repeat hairpin DNA helps explain the genetic evidence that Srs2, not the RecQ homolog Sgs1, is a preferred helicase for preventing expansions.     

The Role of XPG in Processing (CAG)n/(CTG)n DNA Hairpins

Background

During DNA replication or repair, disease-associated (CAG)_n/(CTG)_n expansion can result from formation of hairpin structures in the repeat tract of the newly synthesized or nicked DNA strand. Recent studies identified a nick-directed (CAG)_n/(CTG)_n hairpin repair (HPR) system that removes (CAG)_n/(CTG)_n hairpins from human cells via endonucleolytic incisions. Because the process is highly similar to the mechanism by which XPG and XPF endonucleases remove bulky DNA lesions during nucleotide excision repair, we assessed the potential role of XPG in conducting (CAG)_n/(CTG)_n HPR.

Results

To determine if the XPG endonuclease is involved in (CAG)_n/(CTG)_n hairpin removal, two XPG-deficient cell lines (GM16024 and AG08802) were examined for their ability to process (CAG)_n/(CTG)_n hairpins in vitro. We demonstrated that the GM16024 cell line processes all hairpin substrates as efficiently as HeLa cells, and that the AG08802 cell line is partially defective in HPR. Analysis of repair intermediates revealed that nuclear extracts from both XPG-deficient lines remove CAG/CTG hairpins via incisions, but the incision products are distinct from those generated in HeLa extracts. We also show that purified recombinant XPG protein greatly stimulates HPR in XPG-deficient extracts by promoting an incision 5' to the hairpin.

Conclusions

Our results strongly suggest that 1) human cells possess multiple pathways to remove (CAG)_n/(CTG)_n hairpins located in newly synthesized (or nicked) DNA strand; and 2) XPG, although not essential for (CAG)_n/(CTG)_n hairpin removal, stimulates HPR by facilitating a 5' incision to the hairpin. This study reveals a novel role for XPG in genome-maintenance and implicates XPG in diseases caused by trinucleotide repeat expansion.     

A small unstructured nucleic acid disrupts a trinucleotide repeat hairpin

A variety of neurodegenerative disorders are associated with the expansion of trinucleotide repeat (TNR) sequences. These repetitive sequences are prone to adopting non-canonical structures, such as intrastrand stem-loop hairpins. Indeed, the formation and persistence of these hairpins during DNA replication and/or repair have been proposed as factors that facilitate TNR expansion. Given this proposed contribution of TNR hairpins to the expansion mechanism, disruption of such structures via strand invasion offers a potential means to negate the disease-initiating expansion. In this work, we investigated the strand invading abilities of a (CTG)₃ unstructured nucleic acid on a (CAG)₁₀ TNR hairpin. Using fluorescence, optical, and electrophoretic methods, instantaneous disruption of the (CAG)₁₀ hairpin by (CTG)₃ was observed at low temperatures. Additionally, we have identified three distinct duplex-like species that form between (CAG)₁₀ and (CTG)₃; these include 1, 2, or 3 (CTG)₃ sequences hybridized to (CAG)₁₀. The results presented here showcase (CTG)₃ as an invader of a TNR hairpin and suggest that unstructured nucleic acids could serve as a scaffold to design agents to prevent TNR expansion.     

In vitro repair of DNA hairpins containing various numbers of CAG/CTG trinucleotide repeats

Expansion of CAG/CTG trinucleotide repeats (TNRs) in humans is associated with a number of neurological and neurodegenerative disorders including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism leads to TNR instability. However, the molecular mechanism by which cells recognize and repair CAG/CTG hairpins is largely unknown. Recent studies have identified a novel DNA repair pathway specifically removing (CAG)(n)/(CTG)(n) hairpins, which is considered a major mechanism responsible for TNR instability. The hairpin repair (HPR) system targets the repeat tracts for incisions in the nicked strand in an error-free manner. To determine the substrate spectrum of the HPR system and its ability to process smaller hairpins, which may be the intermediates for CAG/CTG expansions, we constructed a series of CAG/CTG hairpin heteroduplexes containing different numbers of repeats (from 5 to 25) and examined their repair in human nuclear extracts. We show here that although repair efficiencies differ slightly among these substrates, removal of the individual hairpin structures all involve endonucleolytic incisions within the repeat tracts in the nicked DNA strand. Analysis of the repair intermediates defined specific incision sites for each substrate, which were all located within the repeat regions. Mismatch repair proteins are not required for, nor do they inhibit, the processing of smaller hairpin structures. These results suggest that the HPR system ensures CAG/CTG stability primarily by removing various sizes of (CAG)(n)/(CTG)(n) hairpin structures during DNA metabolism.     

Influence of sequence context and length on the structure and stability of triplet repeat DNA oligomers

Genetic expansion diseases have been linked to the properties of triplet repeat DNA sequences during replication. The most common triplet repeats associated with such diseases are CAG, CCG, CGG, and CTG. It has been suggested that gene expansion occurs as a result of hairpin formation of long stretches of these sequences on the leading daughter strand synthesized during DNA replication [Gellibolian, R., Bacolla, A., and Wells, R. D. (1997) J. Biol. Chem. 272, 16793-7]. To test the biophysical basis for this model, oligonucleotides of general sequence (CNG)(n), where N = A, C, G, or T and n = 4, 5, 10, 15, or 25, were synthesized and characterized by circular dichroism (CD) spectropolarimetry, optical melting studies, and differential scanning calorimetry (DSC). The goal of these studies was to evaluate the influence of sequence context and oligomer length on their secondary structures and stabilities. The results indicate that all single oligomers, even those as short as 12 nucleotides, form stable hairpin structures at 25 degrees C. Such hairpins are characterized by the presence of N:N mismatched base pairs sandwiched between G:C base pairs in the stems and loops of three to four unpaired bases. Thermodynamic analysis of these structures reveals that their stabilities are influenced by both the sequence of the particular oligomer and its length. Specifically, the stability order of CGG > CTG > CAG > CCG was observed. In addition, longer oligomers were found to be more stable than shorter oligomers of the same sequence. However, a stability plateau above 45 nucleotides suggests that the length dependence reaches a maximum value where the stability of the G:C base pairs can no longer compensate the instability of the N:N mismatches in the stems of the hairpins. The results are discussed in terms of the above model proposed for gene expansion.     

Base excision repair of oxidative DNA damage coupled with removal of a CAG repeat hairpin attenuates trinucleotide repeat expansion

Trinucleotide repeat (TNR) expansion is responsible for numerous human neurodegenerative diseases. However, the underlying mechanisms remain unclear. Recent studies have shown that DNA base excision repair (BER) can mediate TNR expansion and deletion by removing base lesions in different locations of a TNR tract, indicating that BER can promote or prevent TNR expansion in a damage location–dependent manner. In this study, we provide the first evidence that the repair of a DNA base lesion located in the loop region of a CAG repeat hairpin can remove the hairpin, attenuating repeat expansion. We found that an 8-oxoguanine located in the loop region of CAG hairpins of varying sizes was removed by OGG1 leaving an abasic site that was subsequently 5′-incised by AP endonuclease 1, introducing a single-strand breakage in the hairpin loop. This converted the hairpin into a double-flap intermediate with a 5′- and 3′-flap that was cleaved by flap endonuclease 1 and a 3′-5′ endonuclease Mus81/Eme1, resulting in complete or partial removal of the CAG hairpin. This further resulted in prevention and attenuation of repeat expansion. Our results demonstrate that TNR expansion can be prevented via BER in hairpin loops that is coupled with the removal of TNR hairpins.     

The purine-rich trinucleotide repeat sequences d(CAG)15 and d(GAC)15 form hairpins.

The structures of single-stranded (ss) oligonucleotides containing (CAG)15 [ss(CAG)15] or (GAC)15 [ss(GAC)15] were examined. At 10 degrees C, the electrophoretic mobilites of the two DNAs were similar to ss(CTG)15, a DNA that forms a hairpin containing base paired and/or stacked thymines. At 37 degrees C in 50 mM NaCl, single-strand-specific P1 nuclease cleaved the G33-G36 phosphodiesters of ss(GAC)15, and the G32-A34, G35-C36 phosphodiesters of ss(CAG)15 (where the loop apex of both DNAs = A34). Electrophoretic mobility melting profiles indicated that the melting temperature (Tm) of ss(CAG)15 in low (approximately 1 mM Na+) ionic strength was 38 degrees C. In contrast, the Tm of ss(GAC)15 was 49 degrees C, a value similar to the Tm of ss(CTG)15. These results provide evidence that ss(GAC)15 and ss(CAG)15 form similar, but distinguishable hairpin structures.     

The Werner Syndrome Protein Promotes CAG/CTG Repeat Stability by Resolving Large (CAG)n/(CTG)n Hairpins

Expansion of CAG/CTG repeats causes certain neurological and neurodegenerative disorders, and the formation and subsequent persistence of stable DNA hairpins within these repeats are believed to contribute to CAG/CTG repeat instability. Human cells possess a DNA hairpin repair (HPR) pathway, which removes various (CAG)(n) and (CTG)(n) hairpins in a nick-directed and strand-specific manner. Interestingly, this HPR system processes a (CTG)(n) hairpin on the template DNA strand much less efficiently than a (CAG)(n) hairpin on the same strand (Hou, C., Chan, N. L., Gu, L., and Li, G. M. (2009) Incision-dependent and error-free repair of (CAG)(n)/(CTG)(n) hairpins in human cell extracts. Nat. Struct. Mol. Biol. 16, 869-875), suggesting the involvement of an additional component for (CTG)(n) HPR. To identify this activity, a functional in vitro HPR assay was used to screen partially purified HeLa nuclear fractions for their ability to stimulate (CTG)(n) HPR. We demonstrate here that the stimulating activity is the Werner syndrome protein (WRN). Although WRN contains both a 3'→5' helicase activity and a 3'→5' exonuclease activity, the stimulating activity was found to be the helicase activity, as a WRN helicase mutant failed to enhance (CTG)(n) HPR. Consistently, WRN efficiently unwound large (CTG)(n) hairpins and promoted DNA polymerase δ-catalyzed DNA synthesis using a (CTG)(n) hairpin as a template. We, therefore, conclude that WRN stimulates (CTG)(n) HPR on the template DNA strand by resolving the hairpin so that it can be efficiently used as a template for repair or replicative synthesis.     

DNA CTG triplet repeats involved in dynamic mutations of neurologically related gene sequences form stable duplexes.

DNA triplet repeats, 5'-d(CTG)n and 5'-d(CAG)n, are present in genes which have been implicated in several neurodegenerative disorders. To investigate possible stable structures formed by these repeating sequences, we have examined d(CTG)n, d(CAG)n and d(CTG).d(CAG)n (n = 2 and 3) using NMR and UV optical spectroscopy. These studies reveal that single stranded (CTG)n (n > 2) forms stable, antiparallel helical duplexes, while the single stranded (CAG)n requires at least three repeating units to form a duplex. NMR and UV melting experiments show that the Tm increases in the order of [(CAG)3]2 < [(CTG)3]2 << (CAG)3.(CTG)3. The (CTG)3 duplex is stable and exhibits similar NMR spectra in solutions containing 0.1-4 M NaCl and at a pH range from 4.6 to 8.8. The (CTG)3 duplex, which contains multiple-T.T mismatches, displays many NMR spectral characteristics similar to those of B-form DNA. However, unique NOE and 1H-31P coupling patterns associated with the repetitive T.T mismatches in the CTG repeats are discerned. These results, in conjunction with recent in vitro studies suggest that longer CTG repeats may form hairpin structures, which can potentially cause interruption in replication, leading to dynamic expansion or deletion of triplet repeats.     

Genetic instabilities in (CTG.CAG) repeats occur by recombination.

The expansion of triplet repeat sequences (TRS) associated with hereditary neurological diseases is believed from prior studies to be due to DNA replication. This report demonstrates that the expansion of (CTG.CAG)(n) in vivo also occurs by homologous recombination as shown by biochemical and genetic studies. A two-plasmid recombination system was established in Escherichia coli with derivatives of pUC19 (harboring the ampicillin resistance gene) and pACYC184 (harboring the tetracycline resistance gene). The derivatives contained various triplet repeat inserts ((CTG.CAG), (CGG.CCG), (GAA.TTC), (GTC.GAC), and (GTG.CAC)) of different lengths, orientations, and extents of interruptions and a control non-repetitive sequence. The availability of the two drug resistance genes and of several unique restriction sites on the plasmids enabled rigorous genetic and biochemical analyses. The requirements for recombination at the TRS include repeat lengths >30, the presence of CTG.CAG on both plasmids, and recA and recBC. Sequence analyses on a number of DNA products isolated from individual colonies directly demonstrated the crossing-over and expansion of the homologous CTG.CAG regions. Furthermore, inversion products of the type [(CTG)(13)(CAG)(67)].[(CTG)(67)(CAG)(13)] were isolated as the apparent result of "illegitimate" recombination events on intrahelical pseudoknots. This work establishes the relationships between CTG.CAG sequences, multiple fold expansions, genetic recombination, formation of new recombinant DNA products, and the presence of both drug resistance genes. Thus, if these reactions occur in humans, unequal crossing-over or gene conversion may also contribute to the expansions responsible for anticipation associated with several hereditary neurological syndromes.     

Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion

The repair protein 8-oxo-7,8-dihydroguanine glycosylase (OGG1) initiates base excision repair (BER) in mammalian cells by removing the oxidized base 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Interestingly, OGG1 has been implicated in somatic expansion of the trinucleotide repeat (TNR) sequence CAG/CTG. Furthermore, a 'toxic oxidation cycle' has been proposed for age-dependent expansion in somatic cells. In this cycle, duplex TNR DNA is (1) oxidized by endogenous species; (2) BER is initiated by OGG1 and the DNA is further processed by AP endonuclease 1 (APE1); (3) a stem-loop hairpin forms during strand-displacement synthesis by polymerase β (pol β); (4) the hairpin is ligated and (5) incorporated into duplex DNA to generate an expanded CAG/CTG region. This expanded region is again subject to oxidation and the cycle continues. We reported previously that the hairpin adopted by TNR repeats contains a hot spot for oxidation. This finding prompted us to examine the possibility that the generation of a hairpin during a BER event exacerbates the toxic oxidation cycle due to accumulation of damage. Therefore, in this work we used mixed-sequence and TNR substrates containing a site-specific 8-oxoG lesion to define the kinetic parameters of human OGG1 (hOGG1) activity on duplex and hairpin substrates. We report that hOGG1 activity on TNR duplexes is indistinguishable from a mixed-sequence control. Thus, BER is initiated on TNR sequences as readily as non-repetitive DNA in order to start the toxic oxidation cycle. However, we find that for hairpin substrates hOGG1 has reduced affinity and excises 8-oxoG at a significantly slower rate as compared to duplexes. Therefore, 8-oxoG is expected to accumulate in the hairpin intermediate. This damage-containing hairpin can then be incorporated into duplex, resulting in an expanded TNR tract that now contains an oxidative lesion. Thus, the cycle restarts and the DNA can incrementally expand.     

Mismatch Recognition Protein MutS�� Does Not Hijack (CAG)n Hairpin Repair in Vitro

CAG repeats form stable hairpin structures, which are believed to be responsible for CAG repeat expansions associated with certain human neurological diseases. Human cells possess an accurate DNA hairpin repair system that prevents expansion of disease-associated CAG repeats. Based on transgenic animal studies, it is suggested that (CAG)_n expansion is caused by abnormal binding of the MutSβ mismatch recognition protein to (CAG)_n hairpins, leading to hijacking mismatch repair function during (CAG)_n hairpin repair. We demonstrate here that MutSβ displays identical biochemical and biophysical activities (including ATP-provoked conformational change, ATPase, ATP binding, and ADP binding) when interacting with a (CAG)_n hairpin and a mismatch. More importantly, our in vitro functional hairpin repair assays reveal that excess MutSβ does not inhibit (CAG)_n hairpin repair in HeLa nuclear extracts. Evidence presented here provides a novel view as to whether or not MutSβ is involved in CAG repeat instability in humans.Expansion of trinucleotide repeats (TNRs)³ causes hereditary neurological disorders such as Huntington disease and myotonic dystrophy, whose clinical symptoms are directly linked to expansion of CAG and CTG repeats, respectively (1–3). The precise mechanisms by which TNR expansion occurs and the factors that promote it are not fully understood. It has been proposed that CAG and CTG repeats form thermostable hairpins that include A-A and T-T mispairs in the hairpin stem (4, 5). Therefore, cellular mechanisms that process DNA hairpin/loop structures and/or A-A or T-T mispairs may influence TNR stability.Recent studies have identified and characterized a DNA hairpin repair (HPR) system in human cells that promotes CAG/CTG repeat stability (6, 7). The mechanism of human HPR involves incision and removal of CAG/CTG repeat hairpins in a nick-directed and proliferating cell nuclear antigen-dependent manner, followed by DNA resynthesis using the continuous strand as a template (6). In addition to human HPR, the human mismatch repair (MMR) system is well known for its role in stabilizing simple repetitive sequences called microsatellites, which are prone to forming small loops or insertion/deletion (ID) mispairs. In human cells, MutSα (MSH2–MSH6) and MutSβ (MSH2–MSH3) both bind to 1–2-nt ID mispairs, but MutSβ has higher affinity for these small loops (8). Defects in MMR genes cause microsatellite instability and predisposition to cancer (9), demonstrating that MMR is essential for genetic stability in human cells. Surprisingly, genetic studies in mice suggest that MutSβ promotes (CAG)_n expansion and TNR instability. These studies show that expansion of a heterologous (CAG)_n tract occurs in wild type and MSH6^−/− mice but that expansion of the (CAG)_n tract is suppressed in MSH2^−/− and MSH3^−/− mice (10, 11). Recently, Owens et al. (11) reported that binding to a (CAG)_n hairpin influences the protein conformation, nucleotide binding, and hydrolysis activities of MutSβ so that they are different from what has been reported for MutSα during mismatch recognition. It is therefore hypothesized that (CAG)_n hairpins, through their ability to alter the biochemical properties of MutSβ, hijack the MMR process, leading to CAG repeat expansion instead of CAG hairpin removal (11). However, it is not clear why MMR, a major genome maintenance system, would promote TNR instability instead of TNR stability. We, therefore, have developed a novel functional assay and examined the validity of this hypothesis. Our results reveal that MutSβ displays normal biochemical activities when binding to CAG hairpins and does not inhibit (CAG)_n hairpin repair. The observations presented here provide novel thoughts on whether or not or how MutSβ is involved in CAG repeat instability in human cells.     

Extra stable 2',5'-linked RNA loops

We prepared hairpins that differ in the connectivity of phosphodiester linkages in the loop (RNA vs 2', 5'-RNA). We find that the stability of the extra stable RNA hairpin 5'-rGGAC(UUCG)GUCC-3' is the same as that observed for the hairpin containing a 2',5'RNA loop, i.e. 5'-rGGAC(UUCG)GUCC-3' (where UUCG = U2'p5'U2'p5' C2'p5'G2'p5'). Also significant is the finding that when the stem is duplex DNA, duplex 2',5'-RNA, or DNA:2',5'-RNA, hairpins with the UUCG loop are more stable than those with UUCG loop.     

The trinucleotide repeat sequence d(GTC)15 adopts a hairpin conformation.

The structure of a single-stranded (ss) oligonucleotide containing (GTC)15 [ss(GTC)15] was examined. As a control, parallel studies were performed with ss(CTG)15, an oligonucleotide that forms a hairpin. Electrophoretic mobility, KMnO4 oxidation and P1 nuclease studies demonstrate that, similar to ss(CTG)15, ss(GTC)15 forms a hairpin containing base paired and/or stacked thymines in the stem. Electrophoretic mobility melting profiles performed in approximately 1 mM Na+ revealed that the melting temperature of ss(GTC)15 and ss(CTG)15 were 38 and 48 degrees C respectively. The loop regions of ss(GTC)15 and ss(CTG)15 were cleaved by single-strand-specific P1 nuclease at the T25-C29 and G26-C27 phosphodiester bonds respectively (where the loop apex of the DNAs is T28). Molecular dynamics simulations suggested that in ss(GTC)15 the loop was bent towards the major groove of the stem, apparently causing an increased exposure of the T25-C29 region to solvent. In ss(CTG)15 guanine--guanine stacking caused a separation of the G26 and C27 bases, resulting in exposure of the intervening phosphodiester to solvent. The results suggest that ss(GTC)15 and ss(CTG)15 form similar, but distinguishable, hairpin structures.