Anna Pluciennik

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.

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 (EcoRIE111Q, 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 EcoRIE111Q 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.

Small Slipped Register Genetic Instabilities in Escherichia coli in Triplet Repeat Sequences Associated with Hereditary Neurological Diseases

Genetic instability investigations on three triplet repeat sequences (TRS) involved in human hereditary neurological diseases (CTG·CAG, CGG·CCG, and GAA·TTC) revealed a high frequency of small expansions or deletions in 3-base pair registers in Escherichia coli. The presence of G to A polymorphisms in the CTG·CAG sequences served as reporters for the size and location of these instabilities. For the other two repeat sequences, length determinations confirmed the conclusions found for CTG·CAG. These studies were conducted in strains deficient in methyl-directed mismatch repair or nucleotide excision repair in order to investigate the involvement of these postreplicative processes in the genetic instabilities of these TRS. The observation that small and large instabilities for (CTG·CAG)175 fall into distinct size classes (1–8 repeats and approximate multiples of 41 repeats, respectively) leads to the conclusion that more than one DNA instability process is involved. The slippage of the complementary strands of the TRS is probably responsible for the small deletions and expansions in methyl-directed mismatch repair-deficient and nucleotide excision repair-deficient cells. A model is proposed to explain the observed instabilities via strand misalignment, incision, or excision, followed by DNA synthesis and ligation. This slippage-repair mechanism may be responsible for the small expansions in type 1 hereditary neurological diseases involving polyglutamine expansions. Furthermore, these observations may relate to the high frequency of small deletionsversus a lower frequency of large instabilities observed in lymphoblastoid cells from myotonic dystrophy patients.

DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli.

The influence of mutations in the 3′ to 5′ exonucleolytic proofreading ε-subunit of Escherichia coliDNA polymerase III on the genetic instabilities of the CGG·CCG and the CTG·CAG repeats that cause human hereditary neurological diseases was investigated. The dnaQ49 ts and themutD5 mutations destabilize the CGG·CCG repeats. The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process. The CTG·CAG repeats were destabilized by thednaQ49 ts mutation by a process mediated by long hairpin loop structures (≥5 repeats). The mutD5 mutator strain stabilized the (CTG·CAG)175 tract, which contained two interruptions. Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains. Shorter uninterrupted tracts expand readily in themutD5 strain, presumably due to the greater stability of long CTG·CAG tracts (>100 repeats) in this strain. When parallel studies were conducted in minimal medium, where the mutD5strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG·CAG and CGG·CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication.

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 MutSβ 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.

MutLα and Proliferating Cell Nuclear Antigen Share Binding Sites on MutSβ

MutSβ (MSH2-MSH3) mediates repair of insertion-deletion heterologies but also triggers triplet repeat expansions that cause neurological diseases. Like other DNA metabolic activities, MutSβ interacts with proliferating cell nuclear antigen (PCNA) via a conserved motif (QXX(L/I)XXFF). We demonstrate that MutSβ-PCNA complex formation occurs with an affinity of ∼0.1 μm and a preferred stoichiometry of 1:1. However, up to 20% of complexes are multivalent under conditions where MutSβ 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 MutSβ not only abolishes PCNA binding, but unlike MutSα, also dramatically attenuates MutSβ-MutLα interaction, MutLα endonuclease activation, and bidirectional mismatch repair. As predicted by these findings, PCNA competes with MutLα for binding to MutSβ, an effect that is blocked by the cell cycle regulator p21CIP1. We propose that MutSβ-MutLα 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 MutSβ- and MutSα-initiated repair events differ in fundamental ways.

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 MutSβ-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 MutSβ 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 MutSβ-, 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 MutSβ- 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.

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

Background: Polyglutamine-expanded androgen receptor forms nuclear inclusions of cleaved AR. Results: Soluble aggregates and insoluble inclusions of polyglutamine-expanded AR contain full-length AR, which becomes proteolyzed by the proteasome within maturing inclusions. Conclusion: Proteolysis of the mutant AR is a late event in pathogenesis. Significance: Therapeutic strategies for SBMA should target pathogenic events involving full-length AR protein. 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.