Site-specific mutagenesis in a homogeneous polyglutamine tract: application to spinocerebellar ataxin-3

Yu Wai Chen

Centre for Protein Engineering and Cambridge University Chemical Laboratory, MRC Centre, Hills Road, Cambridge CB2 2QH, UK.E-mail: ywc{at}cantab.net


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent years, nine neurodegenerative diseases have been found to be caused by the expansion of a CAG-triplet repeat in the coding region of the respective genes, resulting in lengthening of an otherwise harmless polyglutamine tract in the gene products. To facilitate structural studies of these disease mechanisms, a general protocol is described that allows site-specific mutations to be introduced into the polyglutamine tract. Based on ‘cassette mutagenesis’, this protocol involves engineering unique restriction sites into the flanking regions of the CAG repeat and subsequently replacing the wild-type CAG repeat with a double-stranded synthetic DNA fragment containing the desired mutations. This method was applied to the spinocerebellar ataxin-3 protein, such that the wild-type amino acid sequence –Q3KQ22 was replaced by a –Q9CQ9– sequence. In this case, the incorporated cysteine residue can be exploited for various chemical modifications, lending the host glutamine repeat to many structural and biophysical techniques for the resolution of a specific residue. The method reported here bypasses many problems that can arise from PCR-based mutagenesis methods.

Keywords: ataxia-3/ataxin-3/CAG triplet/Machado–Joseph disease/MJD/mutagenesis/polyglutamine/SCA3


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Over the past decade, a growing number of inherited neurodegenerative diseases, including Huntington’s disease and several forms of spinocerebellar ataxia, have been shown to be related to a polymorphic CAG-triplet repeat motif inside the coding region of the respective gene. Abnormal expansion beyond a threshold number of 35–40 triplets translates into lengthening of the otherwise harmless polyglutamine tract. Although the proteins involved are mutually unrelated, these diseases share several common features and appear to progress via a similar pathogenic mechanism. Expansion of the glutamine repeat confers upon the disease protein a toxic gain of function that causes neuronal death in a tissue-specific manner. In order to understand the structural basis of cytotoxicity, a method of introducing amino acid mutations targeted to specific locations within the polyglutamine tract is desirable.

It is technically challenging to perform site-directed mutagenesis in a CAG repeat, using current mutagenesis methods based on the polymerase chain reaction (PCR). The major problem is that the mutation site cannot be anchored in a homogeneous repeat region. If one uses very long primers that include flanking sequences, other problems such as deletions may arise in the homogeneous CAG repeat during cloning. Here a non-PCR mutagenesis protocol is described that can be generally applied to all nine cases of polyglutamine-expansion diseases. This protocol was successfully applied to generate a site-specific mutant of spinocerebellar ataxin-3.

The gene responsible for spinocerebellar ataxia-3 (SCA3), also known as Machado–Joseph disease (MJD), was cloned in 1994 (Kawaguchi et al., 1994Go). In the MJD1 gene cloned from a normal individual, the translated amino acid sequence corresponds to a protein of 360 amino acids with a glutamine repeat containing 25 glutamines (Q) interrupted by one lysine (K), i.e. –Q3KQ22–. The gene contains a homogeneous repeat of (CAG)20. The primary objective of this work was to create an MJD mutant with a single amino acid replacement from a specific glutamine to a cysteine residue, which can then be exploited for many structural techniques based on cysteine chemistry.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Design of unique restriction sites

The coding DNA sequences of the following proteins were obtained from the SWISS-PROT database (http://www.ebi.ac.uk/swissprot), namely, Huntington’s disease protein (Htt, accession number P42858), androgen receptor (AR, P10275), atrophin-1 (Atf1, P54259), spinocerebellar ataxin-1 (Atx1, P54253), ataxin-2 (Atx2, Q99700), ataxin-3 (Atx3 or MJD, P54252), ataxin-6 (Atx6, O00555), ataxin-7 (Atx7, O15265) and ataxin-17 (Atx17, also known as TATA-box binding protein, TBP or TF2D, P20226). In designing unique restriction sites, it was assumed that each gene was cloned as a BamHI–HindIII fragment into the pRSET A vector (Invitrogen) for sequence analysis. All the gene sequences analysed contain non-pathogenic numbers of CAG repeats. For the Htt protein, only the corresponding exon 1 DNA sequence is analysed.

Elimination of BstBI site in the GST gene

This was achieved by introducing a silent mutation at a phenylalanine codon (TTC->TTT) by a method based on the QuikChange mutagenesis protocol (Stratagene), using a pair of completely complementary mutagenic primers: 5'-CCTGAAATGCTGAAAATGTTTGAAGATCGTTTATGTC-3' and 5'-GACATAAACGATCTTCAAACATTTTCAGCATTTCAGG-3'. Amplification was carried out in 50 µl of a mixture including 200 ng of template pGEX-MJDFL, 5 pmol of each primer, 200 µM of each dNTP, 1.3 U of cloned Pfu DNA polymerase (Stratagene) in 1x cloned Pfu DNA polymerase buffer (Stratagene) and 10% dimethyl sulphoxide (DMSO). The thermal cycler was programmed as follows: initial denaturation at 94°C for 1 min; 30 cycles at 94°C for 10 s, 65°C (-1°C/cycle) for 1 min and 68°C for 5 min; and a final extension at 72°C for 10 min. After the amplification, 1 µl (20 U) of DpnI (Stratagene) was added to the mixture and incubated at 37°C for 1 h. A 1.5 µl volume of the amplified product was then used to transform 50 µl of subcloning-grade XL1-Blue competent cells (Stratagene). This construct is called pG{Delta}.

Introducing a new BstBI site into the MJD1 gene

A new BstBI site was created with a two-step PCR megaprimer mutagenesis method (Kammann et al., 1989Go) employing three primers with the modification of Datta (1995)Go. For the first PCR reaction, we used the sense mutagenic primer 5'-CGGAAGAGACGAGAAGCCTACTTCGAAAAACAGC-3' and the antisense primer 5'-CCGGGAGCTGCATGTGTCAGAGG-3'. PCR was carried out in a 50 µl reaction including 50 ng of template pGEX-MJDFL, 5 pmol of each primer, 200 µM of each dNTP, 1.3 U of cloned Pfu DNA polymerase in 1x AM buffer (Mangiarini et al., 1996Go) with 10% DMSO and 1 mg/ml bovine serum albumin (BSA). The thermal cycler program for amplification was as follows: denaturation at 94°C for 5 min; 25 cycles at 94°C for 1 min, 72°C (-1°C/cycle) for 1 min and 72°C for 2 min; followed by a post-incubation at 72°C for 10 min. The ~300 bp PCR product was purified with EZ enzyme remover (Millipore) and Microcon 50 (Millipore).

The second asymmetric PCR reaction was performed with the sense primer 5'-GAGCGCGATGAAGGTGATAAATGGCGAAAC-3' and the megaprimer which is the major product of the first PCR. In a 50 µl reaction, the following mixture was prepared: 50 ng of template pGEX-MJDFL, 20 µl (out of the 50 µl after Microcon concentration) of the first PCR product (the megaprimer), 200 µM of each dNTP, 1.3 U of cloned Pfu DNA polymerase in 1x cloned Pfu DNA polymerase buffer with 10% DMSO. Five cycles of linear amplification with the megaprimers were performed before the sense primer was added. The thermal cycler was programmed as follows: denaturation at 94°C for 5 min; five linear amplification cycles at 94°C for 1 min and 72°C for 3 min; the sense primer was added at the end of the last 72°C extension step, then 25 cycles at 94°C for 1 min, 60°C for 1 min and 72°C for 3 min; followed by a final extension at 72°C for 10 min. The second PCR product, a 1.6 kb fragment, was purified with EZ enzyme remover and Microcon 50.

The purified DNA, containing the modified MJD1 gene, was cloned into pG{Delta} (see previous section) by exchanging a 1.1 kb BamHI–NotI fragment. The modified version of pGEX–MJDFL, pG{Delta}M+ (see Figure 2AGo), was used to transform XL1-Blue cells. The DNA prepared was sequenced and confirmed to be containing the unique BstBI site immediately upstream of the CAG repeat.



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Fig. 2. MJD mutagenesis strategy. (A) The vector plasmid, pG{Delta}M+, containing unique restriction sites flanking the CAG repeat. The naturally existing BstBI site (in parentheses) in the GST gene has been eliminated first. (B) Mutagenesis was effected by exchanging the segment of DNA encoding the glutamine repeat (Q3KQ22) by a synthetic fragment encoding Q9CQ9.

 
The plasmid was prepared for fragment exchange by double restriction digestion. A 500 ng amount of the vector was digested first with 1 µl (20 U) of BstBI at 65°C for 1 h in 1x buffer 4 (New England Biolabs) with 1 mg/ml BSA in a 20 µl reaction. After briefly cooling on ice, 1 µl (2 U) of PpuMI was added and the reaction was further incubated at 37°C for 1 h. A 0.5 µl (10 U) amount of alkaline phosphatase (Boehringer Mannheim) was added and the reaction incubated at 37°C for a further 1 h to dephosphorylate the cut ends. The reaction products were analysed with a 0.9% agarose gel and the DNA was purified with a QIAquick gel extraction kit (Qiagen).

Preparation of the insert

A pair of mutually priming oligos, 5'-CGAAAAA(CAA)4-CAG(CAA)2CAG)2TGTCAG(CAA)2CAGCAA(CAG)4CGG-3' and 5'-GTCCCG(CTG)4TTGCTG(TTG)2CTGACA(CTG)2(TTG)2CTG(TTG)4TTTTT-3', were designed such that they are complementary with each other but contain overhangs at the termini to reconstitute the respective cohesive ends (BstBI at 5' and PpuMI at 3') for cloning (Figure 2BGo). The two synthesized oligos were pooled together and phosphorylated with T4 polynucleotide kinase (New England Biolabs) and then subjected to denaturation at 95°C for 5 min followed by slow annealing in steps of -1°C/min down to 15°C in a thermal cycler.

Ligation and transformation

To achieve an insert:vector molar ratio of 10:1, 0.4 pmol of insert and 150 ng (~0.04 pmol) of doubly cut vector DNA were pooled together, dried and then ligated with 0.5 µl (2000 U) of high-concentration T4 DNA ligase (New England Biolabs) in 1x T4 DNA ligase buffer (New England Biolabs). The 10 µl reaction mixture was incubated at room temperature for 3.5 h followed by inactivation at 65°C for 10 min before storing on ice. A 1 µl volume of the ligation reaction was used to transform one aliquot of single-shot NovaBlue competent cells (Novagen). DNA was prepared from the transformation colonies and the success of ligation was confirmed by DNA sequencing.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
General mutagenesis strategy

The strategy presented here is based on ‘cassette mutagenesis’, a method dates back to the mid-1980s (Wells et al., 1985Go), involving restriction digestions and ligation with synthetic DNA fragments. PCR is employed only in preparing unique restriction sites.

The basic principle is to find or engineer unique restriction sites into the sequences flanking the CAG repeat so that a new synthetic repeat containing the desired mutation(s) can be introduced by fragment exchange. The nine genes that are implicated in polyglutamine expansion diseases are analysed and the respective suitable restriction sites are summarized in Figure 1Go. Whenever possible, a unique restriction site is engineered (by silent mutation(s) regarding the gene product) within five codons at either end of the CAG repeat. In several of these genes, the sequence downstream to the CAG repeat has very low complexity (namely in the genes of Htt exon 1, Atx2, Atx7 and Atx17), making it impossible to design suitable unique restriction sites in these regions. The workaround is to insert type IIS restriction enzyme recognition sites between the CAG repeat and its downstream flanking sequence. Type IIS restriction enzymes generate sticky ends downstream of and outside their recognition sequences and thus the artificially inserted recognition motifs will be removed after digestion. The only drawback is that the restriction site will not be reconstituted after ligation of the new insert. This is an acceptable solution to the problem since no further engineering will be necessary after site-specific mutagenesis. By careful design, suitable restriction sites can be identified in all nine disease genes (Figure 1Go). After double restriction digestion by the respective endonucleases, each construct will have two incompatible termini ready to receive a synthetic DNA fragment. The synthesis of the insert is relatively straightforward (see Materials and methods).



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Fig. 1. Unique restriction sites design for CAG-repeat flanking sequences. All constructs are assumed to contain a BamHI–HindIII fragment of the gene cloned into the plasmid pRSET A. For each gene, the sense strand of the wild-type sequence is shown in grey on top with the respective translated amino acid sequence. Point mutations are shown in red and insertions are shown in green. The restriction enzyme recognition sequences are highlighted in boxes and cut sites are indicated with triangles. §Need to knock out (by silent mutation) one other site in the gene or the vector to make this site unique.

 
Application to spinocerebellar ataxin-3

The plasmid pGEX-MJDFL contains the MJD1 gene (Kawaguchi et al., 1994Go) cloned into the BamHI–NotI site of the pGEX-5X-1 vector (Pharmacia) that allows the overproduction of MJD as a fusion with glutathione S-transferase (GST). The first step involved identification of unique restriction sites flanking the CAG repeat, such that the CAG repeat can be replaced by an artificially synthesized insert (Figure 2BGo) with the corresponding sticky ends. In the wild-type MJD1 gene, there is an existing PpuMI restriction site immediately 3' to the repeat (Figures 1Go and 2BGo). In the 5' end, it is most convenient to engineer a BstBI site by a single base silent mutation (TTT->TTC) at the codon encoding for a phenylalanine residue upstream of the repeat. A natural BstBI site in the GST gene of pGEX–MJDFL needs to be eliminated (Figure 2AGo). The two silent mutations resulted in translocation of a BstBI site from the GST gene to the MJD1 gene. This plasmid, pG{Delta}M+, was then subjected to double restriction digestion and the wild-type CAG repeat was replaced with the synthetic repeat. The DNA sequencing results are shown in Figure 3Go, confirming that the synthetic fragment has been incorporated successfully. The overall mutation made was to replace a fragment containing the amino acid sequence of –Q3KQ22– (wild type) with –Q9CQ9– (Figures 2BGo and 3Go).



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Fig. 3. DNA sequencing chromatogram confirmed the success of fragment exchange. The grey boxes at the top are single-letter representations of the amino acids (F, phenylalanine; E, glutamine; K, lysine; Q, glutamine; C, cysteine; R, arginine; D, aspartic acid; L, leucine) encoded by the DNA sequence. The pink box represents the recognition sequence of the respective restriction enzymes and the red triangle indicates the cleavage site.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many problems were experienced when prevailing PCR-based methods were used to introduce mutations in the homogeneous CAG repeat region (results not shown). The major hurdles are that the PCR mutagenic primers cannot be anchored, the possibility of undesirable multiple mutations and that insertions and deletions are likely to occur in the repeat region during the PCR cycles.

It is shown here that site-specific mutations can be engineered into a polyglutamine tract by cassette mutagenesis and this method is applicable to all nine known cases of polyglutamine-expansion diseases. This is an important step towards delineating the pathogenic mechanism of these neurodegenerative disorders. At the moment, knowledge of the biochemical and biophysical properties of these proteins remains scarce. Very little is known about the structure of the glutamine repeat in these proteins (Chen et al., 1999Go; Masino et al., 2002Go). Site-specific mutagenesis allows probes (e.g. spin-labels or fluorescence probes) to be introduced into specific locations in the polyglutamine tract and pave the way for residue-resolution structural studies.

In designing the synthetic insert, the intention was to break the homogeneity of the native CAG repeat by introducing irregular combinations of CAA and CAG codons, both coding for glutamine (Figure 3Go). It was hoped that this would open the new construct to downstream PCR methods by enhancing the specificity of anchorage of primers in the repeat sequence and also reduce the risks of insertions and deletions during cloning.

Apparently this method can be adopted to introduce mutations into disease genes with expanded CAG repeats. For this purpose, one would probably need to use several synthetic DNA fragments, e.g. a fragment containing the mutations and some fragments containing the CAA/CAG repeats, and assemble them in a way analogous to gene synthesis.


    Acknowledgments
 
The author is grateful to the late Max Perutz for comments. The plasmid pGEX–MJDFL was a gift from Erich Wanker. This work was supported by Wellcome Trust Grant 061836.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chen,Y.W., Stott,K. and Perutz,M.F. (1999) Proc. Natl Acad. Sci. USA, 96, 1257–1261.[Abstract/Free Full Text]

Datta,A.K. (1995) Nucleic Acids Res., 23, 4530–4531.[ISI][Medline]

Kammann,M., Laufs,J., Schell,J. and Gronenborn,B. (1989) Nucleic Acids Res., 17, 5404.[ISI][Medline]

Kawaguchi,Y. et al. (1994) Nature Genet., 8, 221–228.[ISI][Medline]

Mangiarini,L. et al. (1996) Cell, 87, 493–506.[ISI][Medline]

Masino,L., Kelly,G., Leonard,K., Trottier,Y. and Pastore,A. (2002) FEBS Lett., 513, 267–272.[CrossRef][ISI][Medline]

Wells,J.A., Vasser,M. and Powers,D.B. (1985) Gene, 34, 315–323.[CrossRef][ISI][Medline]

Received May 8, 2002; revised November 6, 2002; accepted December 3, 2002.