FAN1 removes triplet repeat extrusions via a PCNA- and RFC-dependent mechanism.

Human genome-wide association studies have identified FAN1 and several DNA mismatch repair (MMR) genes as modifiers of Huntington's disease age of onset. In animal models, FAN1 prevents somatic expansion of CAG triplet repeats, whereas MMR proteins promote this process. To understand the molecular basis of these opposing effects, we evaluated FAN1 nuclease function on DNA extrahelical extrusions that represent key intermediates in triplet repeat expansion. Here, we describe a strand-directed, extrusion-provoked nuclease function of FAN1 that is activated by RFC, PCNA, and ATP at physiological ionic strength. Activation of FAN1 in this manner results in DNA cleavage in the vicinity of triplet repeat extrahelical extrusions thereby leading to their removal in human cell extracts. The role of PCNA and RFC is to confer strand directionality to the FAN1 nuclease, and this reaction requires a physical interaction between PCNA and FAN1. Using cell extracts, we show that FAN1-dependent CAG extrusion removal relies on a very short patch excision-repair mechanism that competes with MutSß-dependent MMR which is characterized by longer excision tracts. These results provide a mechanistic basis for the role of FAN1 in preventing repeat expansion and could explain the antagonistic effects of MMR and FAN1 in disease onset/progression.

The role of ubiquitination in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative and neuromuscular genetic disease caused by the expansion of a polyglutamine-encoding CAG tract in the androgen receptor (AR) gene. The AR is an important transcriptional regulator of the nuclear hormone receptor superfamily; its levels are regulated in many ways including by ubiquitin-dependent degradation. Ubiquitination is a post-translational modification (PTM) which plays a key role in both AR transcriptional activity and its degradation. Moreover, the ubiquitin-proteasome system (UPS) is a fundamental component of cellular functioning and has been implicated in diseases of protein misfolding and aggregation, including polyglutamine (polyQ) repeat expansion diseases such as Huntington's disease and SBMA. In this review, we discuss the details of the UPS system, its functions and regulation, and the role of AR ubiquitination and UPS components in SBMA. We also discuss aspects of the UPS that may be manipulated for therapeutic effect in SBMA.

DNA Mismatch Repair and its Role in Huntington's Disease.

DNA mismatch repair (MMR) is a highly conserved genome stabilizing pathway that corrects DNA replication errors, limits chromosomal rearrangements, and mediates the cellular response to many types of DNA damage. Counterintuitively, MMR is also involved in the generation of mutations, as evidenced by its role in causing somatic triplet repeat expansion in Huntington's disease (HD) and other neurodegenerative disorders. In this review, we discuss the current state of mechanistic knowledge of MMR and review the roles of key enzymes in this pathway. We also present the evidence for mutagenic function of MMR in CAG repeat expansion and consider mechanistic hypotheses that have been proposed. Understanding the role of MMR in CAG expansion may shed light on potential avenues for therapeutic intervention in HD.

Deubiquitinase USP7 contributes to the pathogenicity of spinal and bulbar muscular atrophy.

Polyglutamine (polyQ) diseases are devastating, slowly progressing neurodegenerative conditions caused by expansion of polyQ-encoding CAG repeats within the coding regions of distinct, unrelated genes. In spinal and bulbar muscular atrophy (SBMA), polyQ expansion within the androgen receptor (AR) causes progressive neuromuscular toxicity, the molecular basis of which is unclear. Using quantitative proteomics, we identified changes in the AR interactome caused by polyQ expansion. We found that the deubiquitinase USP7 preferentially interacts with polyQ-expanded AR and that lowering USP7 levels reduced mutant AR aggregation and cytotoxicity in cell models of SBMA. Moreover, USP7 knockdown suppressed disease phenotypes in SBMA and spinocerebellar ataxia type 3 (SCA3) fly models, and monoallelic knockout of Usp7 ameliorated several motor deficiencies in transgenic SBMA mice. USP7 overexpression resulted in reduced AR ubiquitination, indicating the direct action of USP7 on AR. Using quantitative proteomics, we identified the ubiquitinated lysine residues on mutant AR that are regulated by USP7. Finally, we found that USP7 also differentially interacts with mutant Huntingtin (HTT) protein in striatum and frontal cortex of a knockin mouse model of Huntington's disease. Taken together, our findings reveal a critical role for USP7 in the pathophysiology of SBMA and suggest a similar role in SCA3 and Huntington's disease.

Impaired Nuclear Export of Polyglutamine-Expanded Androgen Receptor in Spinal and Bulbar Muscular Atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by polyglutamine (polyQ) expansion in the androgen receptor (AR). Prior studies have highlighted the importance of AR nuclear localization in SBMA pathogenesis; therefore, in this study, we sought to determine the role of AR nuclear export in the pathological manifestations of SBMA. We demonstrate here that the nuclear export of polyQ-expanded AR is impaired, even prior to the formation of intranuclear inclusions of aggregated AR. Additionally, we find that promoting AR export with an exogenous nuclear export signal substantially reduces its aggregation and blocks hormone-induced toxicity. Moreover, we show that these protective effects are conferred by destabilization of the mutant protein due to an increase in proteasomal degradation of the cytoplasmic AR. Despite a growing body of evidence that global disruption of nucleo/cytoplasmic transport occurs in ALS and HD, our data suggest that no such global disruption occurs in models of SBMA; rather, AR-specific mechanisms, including reduced phosphorylation at Serine 650, are likely responsible for the impaired nuclear export of polyQ-expanded AR.

Preventing the Androgen Receptor N/C Interaction Delays Disease Onset in a Mouse Model of SBMA.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by a polyglutamine expansion in the androgen receptor (AR) and is associated with misfolding and aggregation of the mutant AR. We investigated the role of an interdomain interaction between the amino (N)-terminal FxxLF motif and carboxyl (C)-terminal AF-2 domain in a mouse model of SBMA. Male transgenic mice expressing polyQ-expanded AR with a mutation in the FxxLF motif (F23A) to prevent the N/C interaction displayed substantially improved motor function compared with N/C-intact AR-expressing mice and showed reduced pathological features of SBMA. Serine 16 phosphorylation was substantially enhanced by the F23A mutation; moreover, the protective effect of AR F23A was dependent on this phosphorylation. These results reveal an important role for the N/C interaction on disease onset in mice and suggest that targeting AR conformation could be a therapeutic strategy for patients with SBMA.

Proteasome-mediated proteolysis of the polyglutamine-expanded androgen receptor is a late event in spinal and bulbar muscular atrophy (SBMA) pathogenesis.

Proteolysis of polyglutamine-expanded proteins is thought to be a required step in the pathogenesis of several neurodegenerative diseases. The accepted view for many polyglutamine proteins is that proteolysis of the mutant protein produces a "toxic fragment" that induces neuronal dysfunction and death in a soluble form; toxicity of the fragment is buffered by its incorporation into amyloid-like inclusions. In contrast to this view, we show that, in the polyglutamine disease spinal and bulbar muscular atrophy, proteolysis of the mutant androgen receptor (AR) is a late event. Immunocytochemical and biochemical analyses revealed that the mutant AR aggregates as a full-length protein, becoming proteolyzed to a smaller fragment through a process requiring the proteasome after it is incorporated into intranuclear inclusions. Moreover, the toxicity-predicting conformational antibody 3B5H10 bound to soluble full-length AR species but not to fragment-containing nuclear inclusions. These data suggest that the AR is toxic as a full-length protein, challenging the notion of polyglutamine protein fragment-associated toxicity by redefining the role of AR proteolysis in spinal and bulbar muscular atrophy pathogenesis.

DNA triplet repeat expansion and mismatch repair.

DNA mismatch repair is a conserved antimutagenic pathway that maintains genomic stability through rectification of DNA replication errors and attenuation of chromosomal rearrangements. Paradoxically, mutagenic action of mismatch repair has been implicated as a cause of triplet repeat expansions that cause neurological diseases such as Huntington disease and myotonic dystrophy. This mutagenic process requires the mismatch recognition factor MutSß and the MutLα (and/or possibly MutLγ) endonuclease, and is thought to be triggered by the transient formation of unusual DNA structures within the expanded triplet repeat element. This review summarizes the current knowledge of DNA mismatch repair involvement in triplet repeat expansion, which encompasses in vitro biochemical findings, cellular studies, and various in vivo transgenic animal model experiments. We present current mechanistic hypotheses regarding mismatch repair protein function in mediating triplet repeat expansions and discuss potential therapeutic approaches targeting the mismatch repair pathway.

Extrahelical (CAG)/(CTG) triplet repeat elements support proliferating cell nuclear antigen loading and MutLα endonuclease activation.

MutLα endonuclease can be activated on covalently continuous DNA that contains a MutSα- or MutSß-recognizable lesion and a helix perturbation that supports proliferating cell nuclear antigen (PCNA) loading by replication factor C, providing a potential mechanism for triggering mismatch repair on nonreplicating DNA. Because mouse models for somatic expansion of disease-associated (CAG)n/(CTG)n triplet repeat sequences have implicated both MutSß and MutLα and have suggested that expansions can occur in the absence of replication, we have asked whether an extrahelical (CAG)n or (CTG)n element is sufficient to trigger MutLα activation. (CAG)n and (CTG)n extrusions in relaxed closed circular DNA do in fact support MutSß-, replication factor C-, and PCNA-dependent activation of MutLα endonuclease, which can incise either DNA strand. Extrahelical elements of two or three repeat units are the preferred substrates for MutLα activation, and extrusions of this size also serve as moderately effective sites for loading the PCNA clamp. Relaxed heteroduplex DNA containing a two or three-repeat unit extrusion also triggers MutSß- and MutLα-endonuclease-dependent mismatch repair in nuclear extracts of human cells. This reaction occurs without obvious strand bias at about 10% the rate of that observed with otherwise identical nicked heteroduplex DNA. These findings provide a mechanism for initiation of triplet repeat processing in nonreplicating DNA that is consistent with several features of the model of Gomes-Pereira et al. [Gomes-Pereira M, Fortune MT, Ingram L, McAbney JP, Monckton DG (2004) Hum Mol Genet 13(16):1815-1825]. They may also have implications for triplet repeat processing at a replication fork.

PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair.

MutLα (MLH1-PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLα incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion.

MutLalpha and proliferating cell nuclear antigen share binding sites on MutSbeta.

MutSbeta (MSH2-MSH3) mediates repair of insertion-deletion heterologies but also triggers triplet repeat expansions that cause neurological diseases. Like other DNA metabolic activities, MutSbeta interacts with proliferating cell nuclear antigen (PCNA) via a conserved motif (QXX(L/I)XXFF). We demonstrate that MutSbeta-PCNA complex formation occurs with an affinity of approximately 0.1 microM and a preferred stoichiometry of 1:1. However, up to 20% of complexes are multivalent under conditions where MutSbeta is in molar excess over PCNA. Conformational studies indicate that the two proteins associate in an end-to-end fashion in solution. Surprisingly, mutation of the PCNA-binding motif of MutSbeta not only abolishes PCNA binding, but unlike MutSalpha, also dramatically attenuates MutSbeta-MutLalpha interaction, MutLalpha endonuclease activation, and bidirectional mismatch repair. As predicted by these findings, PCNA competes with MutLalpha for binding to MutSbeta, an effect that is blocked by the cell cycle regulator p21(CIP1). We propose that MutSbeta-MutLalpha interaction is mediated in part by residues ((L/I)SRFF) embedded within the MSH3 PCNA-binding motif. To our knowledge this is the first case where residues important for PCNA binding also mediate interaction with a second protein. These findings also indicate that MutSbeta- and MutSalpha-initiated repair events differ in fundamental ways.

Involvement of the beta clamp in methyl-directed mismatch repair in vitro.

We have examined function of the bacterial beta replication clamp in the different steps of methyl-directed DNA mismatch repair. The mismatch-, MutS-, and MutL-dependent activation of MutH is unaffected by the presence or orientation of loaded beta clamp on either 3' or 5' heteroduplexes. Similarly, beta is not required for 3' or 5' mismatch-provoked excision when scored in the presence of gamma complex or in the presence of gamma complex and DNA polymerase III core components. However, mismatch repair does not occur in the absence of beta, an effect we attribute to a requirement for the clamp in the repair DNA synthesis step of the reaction. We have confirmed previous findings that beta clamp interacts specifically with MutS and MutL (López de Saro, F. J., Marinus, M. G., Modrich, P., and O'Donnell, M. (2006) J. Biol. Chem. 281, 14340-14349) and show that the mutator phenotype conferred by amino acid substitution within the MutS N-terminal beta-interaction motif is the probable result of instability coupled with reduced activity in multiple steps of the repair reaction. In addition, we have found that the DNA polymerase III alpha catalytic subunit interacts strongly and specifically with both MutS and MutL. Because interactions of polymerase III holoenzyme components with MutS and MutL appear to be of limited import during the initiation and excision steps of mismatch correction, we suggest that their significance might lie in the control of replication fork events in response to the sensing of DNA lesions by the repair system.

Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair.

The hemimethylated d(GATC) sequence that directs Escherichia coli mismatch repair can reside on either side of a mismatch at a separation distance of 1,000 bp or more. Initiation of repair involves the mismatch-, MutS-, and MutL-dependent activation of MutH endonuclease, which incises the unmethylated strand at the d(GATC) sequence, with the ensuing strand break serving as the loading site for the appropriate 3'-to-5' or 5'-to-3' excision system. However, the mechanism responsible for the coordinated recognition of the mismatch and a hemimodified d(GATC) site is uncertain. We show that a protein roadblock (EcoRI(E111Q), a hydrolytically defective form of EcoRI endonuclease) placed on the helix between the two DNA sites inhibits MutH activation by 70-80% and that events that escape inhibition are attributable, at least in part, to diffusion of EcoRI(E111Q) away from its recognition site. We also demonstrate that a double-strand break located within the shorter path linking the mismatch and a d(GATC) site in a circular heteroduplex abolishes MutH activation, whereas a double-strand break within the longer path is without effect. These findings support the idea that initiation of mismatch repair involves signaling along the helix contour.

Bacterial expression system with tightly regulated gene expression and plasmid copy number.

A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.

Long CTG.CAG repeat sequences markedly stimulate intramolecular recombination.

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

Long CTG.CAG repeats from myotonic dystrophy are preferred sites for intermolecular recombination.

Homologous recombination was shown to enable the expansion of CTG.CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG.CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG.CAG)(n) repeats apparently recombine with an approximately 60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG.CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG.CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.