Trinucleotide repeat expansions catalyzed by human cell-free extracts

Trinucleotide repeat expansions cause 17 heritable human neurological disorders. In some diseases, somatic expansions occur in non-proliferating tissues such as brain where DNA replication is limited. This finding stimulated significant interest in replication-independent expansion mechanisms. Aberrant DNA repair is a likely source, based in part on mouse studies showing that somatic expansions are provoked by the DNA repair protein MutSβ (Msh2-Msh3 complex). Biochemical studies to date used cell-free extracts or purified DNA repair proteins to yield partial reactions at triplet repeats. The findings included expansions on one strand but not the other, or processing of DNA hairpin structures thought to be important intermediates in the expansion process. However, it has been difficult to recapitulate complete expansions in vitro, and the biochemical role of MutSβ remains controversial. Here, we use a novel in vitro assay to show that human cell-free extracts catalyze expansions and contractions of trinucleotide repeats without the requirement for DNA replication. The extract promotes a size range of expansions that is similar to certain diseases, and triplet repeat length and sequence govern expansions in vitro as in vivo. MutSβ stimulates expansions in the extract, consistent with aberrant repair of endogenous DNA damage as a source of expansions. Overall, this biochemical system retains the key characteristics of somatic expansions in humans and mice, suggesting that this important mutagenic process can be restored in the test tube.     

(CAG)(n)-hairpin DNA binds to Msh2-Msh3 and changes properties of mismatch recognition

Cells have evolved sophisticated DNA repair systems to correct damaged DNA. However, the human DNA mismatch repair protein Msh2-Msh3 is involved in the process of trinucleotide (CNG) DNA expansion rather than repair. Using purified protein and synthetic DNA substrates, we show that Msh2-Msh3 binds to CAG-hairpin DNA, a prime candidate for an expansion intermediate. CAG-hairpin binding inhibits the ATPase activity of Msh2-Msh3 and alters both nucleotide (ADP and ATP) affinity and binding interfaces between protein and DNA. These changes in Msh2-Msh3 function depend on the presence of A.A mispaired bases in the stem of the hairpin and on the hairpin DNA structure per se. These studies identify critical functional defects in the Msh2-Msh3-CAG hairpin complex that could misdirect the DNA repair process.     

Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates

DNA mismatch repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae, mispairs are primarily detected by the Msh2-Msh6 complex and corrected following recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. We found that the Msh2-Msh6 complex is an S phase component of replication centers independent of mispaired bases; this localized pool accounted for 10%-15% of MMR in wild-type cells but was essential for MMR in the absence of Exo1. Unexpectedly, Mlh1-Pms1 formed nuclear foci that, although dependent on Msh2-Msh6 for formation, rarely colocalized with Msh2-Msh6 replication-associated foci. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These findings suggest the presence of replication machinery-coupled and -independent pathways for mispair recognition by Msh2-Msh6, which direct formation of superstoichiometric Mlh1-Pms1 foci that represent sites of active MMR.     

Postreplication repair inhibits CAG.CTG repeat expansions in Saccharomyces cerevisiae

Trinucleotide repeats (TNRs) are unique DNA microsatellites that can expand to cause human disease. Recently, Srs2 was identified as a protein that inhibits TNR expansions in Saccharomyces cerevisiae. Here, we demonstrate that Srs2 inhibits CAG . CTG expansions in conjunction with the error-free branch of postreplication repair (PRR). Like srs2 mutants, expansions are elevated in rad18 and rad5 mutants, as well as the PRR-specific PCNA alleles pol30-K164R and pol30-K127/164R. Epistasis analysis indicates that Srs2 acts upstream of these PRR proteins. Also, like srs2 mutants, the pol30-K127/164R phenotype is specific for expansions, as this allele does not alter mutation rates at dinucleotide repeats, at nonrepeating sequences, or for CAG . CTG repeat contractions. Our results suggest that Srs2 action and PRR processing inhibit TNR expansions. We also investigated the relationship between PRR and Rad27 (Fen1), a well-established inhibitor of TNR expansions that acts at 5' flaps. Our results indicate that PRR protects against expansions arising from the 3' terminus, presumably replication slippage events. This work provides the first evidence that CAG . CTG expansions can occur by 3' slippage, and our results help define PRR as a key cellular mechanism that protects against expansions.     

Cadmium inhibits the functions of eukaryotic MutS complexes

Exposure of yeast cells to low concentrations of cadmium results in elevated mutation rates due to loss of mismatch repair (MMR), and cadmium inhibits MMR activity in extracts of human cells. Here we show that cadmium inhibits both Msh2-Msh6- and Msh2-Msh3-dependent human MMR activity in vitro. This inhibition, which occurs at a step or steps preceding repair DNA synthesis, is observed for repair directed by either a 3' or a 5' nick. In an attempt to identify the protein target(s) of cadmium inhibition, we show that cadmium inhibition of MMR is not reversed by addition of zinc to the repair reaction, suggesting that the target is not a zinc metalloprotein. We then show that cadmium inhibits ATP hydrolysis by yeast Msh2-Msh6 but has no effect on ATPase hydrolysis by yeast Mlh1-Pms1. Steady state kinetic analysis with wild type Msh2-Msh6, and with heterodimers containing subunit-specific Glu to Ala replacements inferred to inactivate the ATPase activity of either Msh2 or Msh6, suggest that cadmium inhibits ATP hydrolysis by Msh6 but not Msh2. Cadmium also reduces DNA binding by Msh2-Msh6 and more so for mismatched than matched duplexes. These data indicate that eukaryotic Msh2-Msh3 and Msh2-Msh6 complexes are targets for inhibition of MMR by cadmium, a human lung carcinogen that is ubiquitous in the environment.     

Multiple factors insulate Msh2-Msh6 mismatch repair activity from defects in Msh2 domain I

DNA mismatch repair (MMR) is a highly conserved mutation avoidance mechanism that corrects DNA polymerase misincorporation errors. In initial steps in MMR, Msh2-Msh6 binds mispairs and small insertion/deletion loops, and Msh2-Msh3 binds larger insertion/deletion loops. The msh2Δ1 mutation, which deletes the conserved DNA-binding domain I of Msh2, does not dramatically affect Msh2-Msh6-dependent repair. In contrast, msh2Δ1 mutants show strong defects in Msh2-Msh3 functions. Interestingly, several mutations identified in patients with hereditary non-polyposis colorectal cancer map to domain I of Msh2; none have been found in MSH3. To understand the role of Msh2 domain I in MMR, we examined the consequences of combining the msh2Δ1 mutation with mutations in two distinct regions of MSH6 and those that increase cellular mutational load (pol3-01 and rad27). These experiments reveal msh2Δ1-specific phenotypes in Msh2-Msh6 repair, with significant effects on mutation rates. In vitro assays demonstrate that msh2Δ1-Msh6 DNA binding is less specific for DNA mismatches and produces an altered footprint on a mismatch DNA substrate. Together, these results provide evidence that, in vivo, multiple factors insulate MMR from defects in domain I of Msh2 and provide insights into how mutations in Msh2 domain I may cause hereditary non-polyposis colorectal cancer.     

MutS�� and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells

Trinucleotide repeat (TNR) expansions cause at least 17 heritable neurological diseases, including Huntington’s disease. Expansions are thought to arise from abnormal processing of TNR DNA by specific trans-acting proteins. For example, the DNA repair complex MutSβ (MSH2–MSH3 heterodimer) is required in mice for on-going expansions of long, disease-causing alleles. A distinctive feature of TNR expansions is a threshold effect, a narrow range of repeat units (∼30–40 in humans) at which mutation frequency rises dramatically and disease can initiate. The goal of this study was to identify factors that promote expansion of threshold-length CTG•CAG repeats in a human astrocytic cell line. siRNA knockdown of the MutSβ subunits MSH2 or MSH3 impeded expansions of threshold-length repeats, while knockdown of the MutSα subunit MSH6 had no effect. Chromatin immunoprecipitation experiments indicated that MutSβ, but not MutSα, was enriched at the TNR. These findings imply a direct role for MutSβ in promoting expansion of threshold-length CTG•CAG tracts. We identified the class II deacetylase HDAC5 as a novel promoting factor for expansions, joining the class I deacetylase HDAC3 that was previously identified. Double knockdowns were consistent with the possibility that MutSβ, HDAC3 and HDAC5 act through a common pathway to promote expansions of threshold-length TNRs.     

DNA base excision repair: a mechanism of trinucleotide repeat expansion

The expansion of trinucleotide repeat (TNR) sequences in human DNA is considered to be a key factor in the pathogenesis of more than 40 neurodegenerative diseases. TNR expansion occurs during DNA replication and also, as suggested by recent studies, during the repair of DNA lesions produced by oxidative stress. In particular, the oxidized guanine base 8-oxoguanine within sequences containing CAG repeats may induce formation of pro-expansion intermediates through strand slippage during DNA base excision repair (BER). In this article, we describe how oxidized DNA lesions are repaired by BER and discuss the importance of the coordinated activities of the key repair enzymes, such as DNA polymerase β, flap endonuclease 1 (FEN1) and DNA ligase, in preventing strand slippage and TNR expansion.     

Effects of sequence on repeat expansion during DNA replication

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

AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair

Base excision repair (BER) of an oxidized base within a trinucleotide repeat (TNR) tract can lead to TNR expansions that are associated with over 40 human neurodegenerative diseases. This occurs as a result of DNA secondary structures such as hairpins formed during repair. We have previously shown that BER in a TNR hairpin loop can lead to removal of the hairpin, attenuating or preventing TNR expansions. Here, we further provide the first evidence that AP endonuclease 1 (APE1) prevented TNR expansions via its 3′-5′ exonuclease activity and stimulatory effect on DNA ligation during BER in a hairpin loop. Coordinating with flap endonuclease 1, the APE1 3′-5′ exonuclease activity cleaves the annealed upstream 3′-flap of a double-flap intermediate resulting from 5′-incision of an abasic site in the hairpin loop. Furthermore, APE1 stimulated DNA ligase I to resolve a long double-flap intermediate, thereby promoting hairpin removal and preventing TNR expansions.     

Mispair-specific Recruitment of the Mlh1-Pms1 Complex Identifies Repair Substrates of the Saccharomyces cerevisiae Msh2-Msh3 Complex

DNA mismatch repair is initiated by either the Msh2-Msh6 or the Msh2-Msh3 mispair recognition heterodimer. Here we optimized the expression and purification of Saccharomyces cerevisiae Msh2-Msh3 and performed a comparative study of Msh2-Msh3 and Msh2-Msh6 for mispair binding, sliding clamp formation, and Mlh1-Pms1 recruitment. Msh2-Msh3 formed sliding clamps and recruited Mlh1-Pms1 on +1, +2, +3, and +4 insertion/deletions and CC, AA, and possibly GG mispairs, whereas Msh2-Msh6 formed mispair-dependent sliding clamps and recruited Mlh1-Pms1 on 7 of the 8 possible base:base mispairs, the +1 insertion/deletion mispair, and to a low level on the +2 but not the +3 or +4 insertion/deletion mispairs and not on the CC mispair. The mispair specificity of sliding clamp formation and Mlh1-Pms1 recruitment but not mispair binding alone correlated best with genetic data on the mispair specificity of Msh2-Msh3- and Msh2-Msh6-dependent mismatch repair in vivo. Analysis of an Msh2-Msh6/Msh3 chimeric protein and mutant Msh2-Msh3 complexes showed that the nucleotide binding domain and communicating regions but not the mispair binding domain of Msh2-Msh3 are responsible for the extremely rapid dissociation of Msh2-Msh3 sliding clamps from DNA relative to that seen for Msh2-Msh6, and that amino acid residues predicted to stabilize Msh2-Msh3 interactions with bent, strand-separated mispair-containing DNA are more critical for the recognition of small +1 insertion/deletions than larger +4 insertion/deletions.     

Saccharomyces cerevisiae Srs2 DNA helicase selectively blocks expansions of trinucleotide repeats

Trinucleotide repeats (TNRs) undergo frequent mutations in families afflicted with certain neurodegenerative disorders and in model organisms. TNR instability is modulated both by the repeat tract itself and by cellular proteins. Here we identified the Saccharomyces cerevisiae DNA helicase Srs2 as a potent and selective inhibitor of expansions. srs2 mutants had up to 40-fold increased expansion rates of CTG, CAG, and CGG repeats. The expansion phenotype was specific, as mutation rates at dinucleotide repeats, at unique sequences, or for TNR contractions in srs2 mutants were not altered. Srs2 is known to suppress inappropriate genetic recombination; however, the TNR expansion phenotype of srs2 mutants was largely independent of RAD51 and RAD52. Instead, Srs2 mainly functioned with DNA polymerase delta to block expansions. The helicase activity of Srs2 was important, because a point mutant lacking ATPase function was defective in blocking expansions. Purified Srs2 was substantially better than bacterial UvrD helicase at in vitro unwinding of a DNA substrate that mimicked a TNR hairpin. Disruption of the related helicase gene SGS1 did not lead to excess expansions, nor did wild-type SGS1 suppress the expansion phenotype of an srs2 strain. We conclude that Srs2 selectively blocks triplet repeat expansions through its helicase activity and primarily in conjunction with polymerase delta.     

Mismatch recognition-coupled stabilization of Msh2-Msh6 in an ATP-bound state at the initiation of DNA repair

Mismatch repair proteins correct errors in DNA via an ATP-driven process. In eukaryotes, the Msh2-Msh6 complex recognizes base pair mismatches and small insertion/deletions in DNA and initiates repair. Both Msh2 and Msh6 proteins contain Walker ATP-binding motifs that are necessary for repair activity. To understand how these proteins couple ATP binding and hydrolysis to DNA binding/mismatch recognition, the ATPase activity of Saccharomyces cerevisiae Msh2-Msh6 was examined under pre-steady-state conditions. Acid-quench experiments revealed that in the absence of DNA, Msh2-Msh6 hydrolyzes ATP rapidly (burst rate = 3 s(-1) at 20 degrees C) and then undergoes a slow step in the pathway that limits catalytic turnover (k(cat) = 0.1 s(-1)). ATP is hydrolyzed similarly in the presence of fully matched duplex DNA; however, in the presence of a G:T mismatch or +T insertion-containing DNA, ATP hydrolysis is severely suppressed (rate = 0.1 s(-1)). Pulse-chase experiments revealed that Msh2-Msh6 binds ATP rapidly in the absence or in the presence of DNA (rate = 0.1 microM(-1) s(-1)), indicating that for the Msh2-Msh6.mismatched DNA complex, a step after ATP binding but before or at ATP hydrolysis is the rate-limiting step in the pathway. Thus, mismatch recognition is coupled to a dramatic increase in the residence time of ATP on Msh2-Msh6. This mismatch-induced, stable ATP-bound state of Msh2-Msh6 likely signals downstream events in the repair pathway.     

Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5′ nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3′ nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg²⁺ and Mn²⁺ for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR.     

Saccharomyces cerevisiae Msh2-Msh3 acts in repair of base-base mispairs

DNA mismatch repair is thought to act through two subpathways involving the recognition of base-base and insertion/deletion mispairs by the Msh2-Msh6 heterodimer and the recognition of insertion/deletion mispairs by the Msh2-Msh3 heterodimer. Here, through genetic and biochemical approaches, we describe a previously unidentified role of the Msh2-Msh3 heterodimer in the recognition of base-base mispairs and the suppression of homology-mediated duplication and deletion mutations. Saccharomyces cerevisiae msh3 mutants did not show an increase in the rate of base substitution mutations by the CAN1 forward mutation assay compared to the rate for the wild type but did show an altered spectrum of base substitution mutations, including an increased accumulation of base pair changes from GC to CG and from AT to TA; msh3 mutants also accumulated homology-mediated duplication and deletion mutations. The mutation spectrum of mlh3 mutants paralleled that of msh3 mutants, suggesting that the Mlh1-Mlh3 heterodimer may also play a role in the repair of base-base mispairs and in the suppression of homology-mediated duplication and deletion mutations. Mispair binding analysis with purified Msh2-Msh3 and DNA substrates derived from CAN1 sequences found to be mutated in vivo demonstrated that Msh2-Msh3 exhibited robust binding to specific base-base mispairs that was consistent with functional mispair binding.     

Genetic instability induced by overexpression of DNA ligase I in budding yeast

Recombination and microsatellite mutation in humans contribute to disorders including cancer and trinucleotide repeat (TNR) disease. TNR expansions in wild-type yeast may arise by flap ligation during lagging-strand replication. Here we show that overexpression of DNA ligase I (CDC9) increases the rates of TNR expansion, of TNR contraction, and of mitotic recombination. Surprisingly, this effect is observed with catalytically inactive forms of Cdc9p protein, but only if they possess a functional PCNA-binding site. Furthermore, in vitro analysis indicates that the interaction of PCNA with Cdc9p and Rad27p (Fen1) is mutually exclusive. Together our genetic and biochemical analysis suggests that, although DNA ligase I seals DNA nicks during replication, repair, and recombination, higher than normal levels can yield genetic instability by disrupting the normal interplay of PCNA with other proteins such as Fen1.     

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.     

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

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

Pms2 Suppresses Large Expansions of the (GAA·TTC)n Sequence in Neuronal Tissues

Expanded trinucleotide repeat sequences are the cause of several inherited neurodegenerative diseases. Disease pathogenesis is correlated with several features of somatic instability of these sequences, including further large expansions in postmitotic tissues. The presence of somatic expansions in postmitotic tissues is consistent with DNA repair being a major determinant of somatic instability. Indeed, proteins in the mismatch repair (MMR) pathway are required for instability of the expanded (CAG·CTG)_n sequence, likely via recognition of intrastrand hairpins by MutSβ. It is not clear if or how MMR would affect instability of disease-causing expanded trinucleotide repeat sequences that adopt secondary structures other than hairpins, such as the triplex/R-loop forming (GAA·TTC)_n sequence that causes Friedreich ataxia. We analyzed somatic instability in transgenic mice that carry an expanded (GAA·TTC)_n sequence in the context of the human FXN locus and lack the individual MMR proteins Msh2, Msh6 or Pms2. The absence of Msh2 or Msh6 resulted in a dramatic reduction in somatic mutations, indicating that mammalian MMR promotes instability of the (GAA·TTC)_n sequence via MutSα. The absence of Pms2 resulted in increased accumulation of large expansions in the nervous system (cerebellum, cerebrum, and dorsal root ganglia) but not in non-neuronal tissues (heart and kidney), without affecting the prevalence of contractions. Pms2 suppressed large expansions specifically in tissues showing MutSα-dependent somatic instability, suggesting that they may act on the same lesion or structure associated with the expanded (GAA·TTC)_n sequence. We conclude that Pms2 specifically suppresses large expansions of a pathogenic trinucleotide repeat sequence in neuronal tissues, possibly acting independently of the canonical MMR pathway.