Long CTG·CAG Repeat Sequences Markedly Stimulate Intramolecular Recombination
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m202128200
ISSN1083-351X
AutoresМарек Напиерала, Paweł Parniewski, Anna Pluciennik, Robert D. Wells,
Tópico(s)Fungal and yeast genetics research
ResumoPrevious 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 ∼2–11 times higher in the recombination proficient strain. Long CTG·CAG tracts recombined ∼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 ∼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. 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 ∼2–11 times higher in the recombination proficient strain. Long CTG·CAG tracts recombined ∼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 ∼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. trinucleotide repeat sequence(s) green fluorescent protein dystrophia myotonica-protein kinase gene Genetic instabilities (expansions and deletions) of simple repeating sequences are important in the life cycles of both prokaryotic (1van Belkum A. Scherer S. van Alphen L. Verbrugh H. Microbiol. Mol. Biol. Rev. 1998; 62: 275-293Crossref PubMed Google Scholar) and eukaryotic (2Wells, R. D., and Warren, S. T. (eds) (1998) Genetic Instabilities and Hereditary Neurological Diseases, Academic Press, San DiegoGoogle Scholar) cells. This fundamental mechanism of mutagenesis has been found in mycoplasma, bacteria, yeast, mammalian cell cultures, and in humans. In mycoplasma and bacteria, these genetic polymorphisms are the basis for phase variations, which control the expression of genes (3De Bolle X. Bayliss C.D. Field D. van de Ven T. Saunders N.J. Hood D.W. Moxon E.R. Mol. 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The general mechanism accepted for all of these instabilities is slipped strand mispairing, which allows mismatching of neighboring repeats and, depending on the strand orientation, enables the insertion or deletion of repeats during DNA polymerase-mediated duplication (reviewed in Refs. 2Wells, R. D., and Warren, S. T. (eds) (1998) Genetic Instabilities and Hereditary Neurological Diseases, Academic Press, San DiegoGoogle Scholar and 13Bowater R.P. Wells R.D. Prog. Nucleic Acids Res. Mol. Biol. 2000; 66: 159-202Crossref Google Scholar). The enzymatic machineries involved include DNA replication and repair (nucleotide excision repair, methyl-directed mismatch repair, and DNA polymerase III proofreading) (2Wells, R. D., and Warren, S. T. (eds) (1998) Genetic Instabilities and Hereditary Neurological Diseases, Academic Press, San DiegoGoogle Scholar, 13Bowater R.P. Wells R.D. Prog. Nucleic Acids Res. Mol. Biol. 2000; 66: 159-202Crossref Google Scholar). Biochemical and genetic studies showed also that expansions and deletions of the TRS1sequences occur in vivo by homologous recombination (14Jakupciak J.P. Wells R.D. J. Biol. Chem. 1999; 274: 23468-23479Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar,15Jakupciak J.P. Wells R.D. J. Biol. Chem. 2000; 275: 40003-40013Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). These investigations, carried out in a two-plasmid system, demonstrated that the expansion mechanism is principally gene conversion rather than unequal crossing-over (15Jakupciak J.P. Wells R.D. J. Biol. Chem. 2000; 275: 40003-40013Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In the present work, we cloned two triplet repeat tracts in the same plasmid and used an intramolecular assay to study the recombinational properties of the CTG·CAG sequences (Fig. 1). 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Evaluation of the frequency of recombination between CTG·CAG tracts provides important information about the cellular mechanisms of instability, relative to replication and repair. Here we have developed the first genetic assay for monitoring the frequencies of intramolecular recombination between CTG·CAG tracts inEscherichia coli. Interestingly, long CTG·CAG repeat sequences from myotonic dystrophy are preferred sites for intramolecular recombination. In our companion paper (27Pluciennik A. Iyer R.R. Napierala M. Larson J.E. Filutowicz M. Wells R.D. J. Biol. Chem. 2002; 277: 34074-34086Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), we have established a genetic assay for monitoring the recombination frequency of the CTG·CAG repeat tracts in an intermolecular system. pRW3244, pRW4026, pRW3246, and pRW3248 were the parent plasmids containing (CTG·CAG)n tracts used for these experiments; these pUC19NotI derivatives contain the (CTG·CAG)n tracts cloned into the HincII site of the polylinker (28Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (315) Google Scholar, 29Pluciennik A. Iyer R.R. Parniewski P. Wells R.D. J. Biol. Chem. 2000; 275: 28386-28397Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 30Bowater R.P. Rosche W.A. Jaworski A. Sinden R.R. Wells R.D. J. Mol. Biol. 1996; 264: 82-96Crossref PubMed Scopus (63) Google Scholar). For nomenclature of the TRS, CTG·CAG designates a duplex sequence of repeating CTG, which may also be written TGC or GCT; CAG, the complementary strand, may also be written as AGC or GCA. The orientation is 5′ to 3′ for both designations of the antiparallel strands. pRW3244 contains (CTG·CAG)17, pRW4026 contains the (CTG·CAG)67, pRW3246 contains (CTG·CAG)98, and pRW3248 contains (CTG·CAG)175 sequence. The (CTG·CAG)175sequence is not a pure CTG·CAG tract but contains two G to A interruptions at repeats 28 and 69 (30); all other TRS are pure (not interrupted). All of these sequences have non-repeating human flanking sequences (19 and 41 bp) outside the repeated tract. pRW3815 2K. Ohshima and R. D Wells, unpublished data. is a pUC18NotI derivative and contains the (GTC·GAC)79 tract. These plasmids were maintained inE. coli HB101 (Invitrogen) (mcrB, mmr, hsdS20(rB−, mB−), recA1, supE44, ara14, galK2, lacY1, proA2, rplS20 (SmR), xyl5, λ−, leuB6, mtl-1). The (CTG·CAG)n and (GTC·GAC)n sequences were subcloned into pBR322. The general strategy of this investigation involved recloning of the (CTG·CAG)n and (GTC·GAC)nsequences from pUC19NotI and pUC18NotI derivatives (28Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (315) Google Scholar), respectively, into pBR322. Fragments containing the CTG·CAG and the GTC·GAC TRS were prepared from these plasmids by digesting the pUC19NotI or pUC18NotI derivatives with EcoRI and HindIII (New England Biolabs, Inc.) followed by filling-in the recessed 3′ termini with 0.1 unit of the Klenow fragment of E. coli DNA polymerase I (U. S. Biochemical Corp.) and the four dNTPs (0.1 mm each). In the case of pRW4806 (a pUC19NotI derivative harboring a tract of 165 uninterrupted CTG·CAG repeats), the insert was prepared byAluI digestion. The blunt-ended DNA fragments were used for cloning to obtain plasmids containing the TRS tracts in both orientations relative to the unidirectional ColE1 origin of replication. The digested DNA was electrophoresed in a 7% polyacrylamide gel and stained with ethidium bromide, and the bands containing the triplet repeat fragment were excised. The DNA was eluted from the excised bands, purified by phenol-chloroform extraction, and precipitated with ethanol (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The vector was prepared by digesting pBR322 with EcoRI and HindIII followed by filling in the recessed 3′ termini as described earlier. The vector and the insert were mixed at a molar ratio of ∼1:10 and ligated for 14 h at 16 °C by the addition of 20 units of T4 DNA ligase (U. S. Biochemical Corp.). The ligation mixture was ethanol-precipitated and transformed into E. coli HB101 by electroporation (2.5 kV, cuvette size 0.2 mm) and plated on LB agar plates containing 100 μg/ml ampicillin. Plasmid DNA was isolated from individual transformants by the Wizard Plus Miniprep DNA Purification System (Promega). Clones containing the CTG·CAG repeats in orientations I and II (defined in Refs. 28Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (315) Google Scholar and 30Bowater R.P. Rosche W.A. Jaworski A. Sinden R.R. Wells R.D. J. Mol. Biol. 1996; 264: 82-96Crossref PubMed Scopus (63) Google Scholar) were obtained and characterized by restriction mapping. The inserts cloned into theEcoRI/HindIII site of pBR322 are referred to as "X inserts" (Fig. 2). The pBR322 derivatives containing a single CTG·CAG sequence (the "X insert") were subsequently used to clone the second TRS tract (CTG·CAG or GTC·GAC) into the PvuII site at position 2064 of the pBR322 backbone (Fig. 2, Y insert). The same experimental approach was used to clone the second TRS insert, except that after ligation the reaction mixture was subjected to PvuII digestion to eliminate plasmids lacking the insert. This strategy enabled the construction of a family of plasmids harboring two homologous TRS tracts oriented as direct repeats or inverted repeats as well as plasmids containing non-homologous repeats (Fig. 2). All plasmids were characterized by restriction mapping (to determine the orientation and length of the cloned TRS) and dideoxy sequencing of both strands with ThermoSequenase Radiolabeled Terminator Cycle Sequencing Kit (U. S. Biochemical Corp.). The sequencing reactions were carried out according to the manufacturer's recommendations using the following pBR322 specific primers: pBR322EcoRI, GTATCACGAGGCCCT which 3′-terminates at the pBR322 map position 4347 (New England Biolabs, Inc.); pBRHR, GCGTTAGCAATTTAACTGTGAT which 3′-terminates at the pBR322 map position 49 (Genosys Inc.); pBRPF, GCTTCACGACCACGCTGAT which 3′-terminates at the pBR322 map position 2052 (Genosys Inc.); pBRPR, GTCAGAGGTTTTCACCGTCAT which 3′-terminates at the pBR322 map position 2087 (Genosys Inc.). The products of the sequencing reactions were analyzed on 6% Long Ranger gels (FMC BioProducts) containing 7.5m urea in the glycerol tolerant gel buffer (U. S. Biochemical Corp.). The gels were dried and exposed to x-ray film. Two different non-repeating sequences were used as controls in this study: the 564-bp fragment of λ phage DNA (HindIII fragment from nucleotide position 36895 to 37459) and the 354-bp fragment of the human DMPK gene (part of the exon 7 and intron 7) (32Mahadevan M. Tsilfidis C. Sabourin L. Shutler G. Amemiya C. Jansen G. Neville C. Narang M. Barcelo¨ J. O'Hoy K. Leblond S. Earle-MacDonald J. de Jong P.J. Wieringa B. Korneluk R.G. Science. 1992; 255: 1253-1255Crossref PubMed Scopus (1422) Google Scholar, 33Fu Y.-H. Pizzuti A. Fenwick R.G., Jr. King J. Rajnarayan S. Dunne P.W. Dubel J. Nasser G.A. Ashizawa T. de Jong P.J. Wieringa B. Korneluk R.G. Perryman M.B. Epstein H.F. Caskey C.T. Science. 1992; 255: 1256-1258Crossref PubMed Scopus (1273) Google Scholar, 34Brook J.D. McCurrach M.E. Harley H.G. Buckler A.J. Church D. Aburatani H. Hunter K. Stanton V.P. Thirion J.P. Hudson T. Sohn R. Zemelman B. Snell R.G. Rundle S.A. Crow S. Davies J. Shelbourne P. Buxton J. Jones C. Juvonen V. 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The PCR primers DM7F, GGCTCGAGACTTCATTCAGC, and DM7R, TAGATGGGCACAGAGCAGGT, were used at the concentration 1 μm. Amplification on a PCR System 9700 (Applied Biosystems) involved 35 cycles: 20 s/95 °C, 20 s/58 °C, and 40 s/72 °C. PAGE-purified PCR product was phosphorylated using 2 mm ATP and 5 units of T4 polynucleotide kinase (New England Biolabs, Inc.) and cloned into the HindIII andPvuII sites of pBR322. pRW4871 as well as pRW4873 contain homologous sequences oriented as direct repeats; however, the orientations of the pairs of inserts are opposite in these two plasmids. pBR322 and pBR322 derivatives containing direct, inverted, non-homologous repeats and non-repeating DNA sequences were digested with EcoRV and EagI (positions 185 and 939 on the pBR322 map, respectively) to remove the 754-bp DNA fragment of the vector backbone. The digested plasmids were purified by 5% acrylamide gel electrophoresis as described earlier and ligated to theGFPuv gene (36Crameri A. Whitehorn E.A. Tate E. Stemmer W.P. Nat. Biotechnol. 1996; 14: 315-319Crossref PubMed Scopus (1053) Google Scholar). The GFPuv gene was obtained by digestion of the pGFPuv (CLONTECH Laboratories, Inc.) withPvuII and EagI (positions 56 and 1078 on the pGFPuv map, respectively). After ligation and transformation intoE. coli HB101, transformants were screened using a long-wave length UV lamp. The cells carrying plasmids with theGFPuv gene emitted a strong green fluorescence. The GFP cassette from pGFPuv contains the GFPuv variant of the green fluorescent protein gene inserted in-frame with the lacZ initiation codon from pUC19 so that a β-galactosidase-GFPuv fusion protein is expressed from the lac promoter in E. coli. For determinations of recombination properties, plasmids containing TRS tracts were electrophoresed in 1% agarose gels, and bands corresponding to the supercoiled plasmids were excised from the gels, transferred into dialysis tubes, and electroeluted (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). To avoid DNA damage, plasmid purifications were performed without ethidium bromide staining and UV irradiation of DNA. In all experiments, only gel-purified, supercoiled plasmid DNA was used for transformation of the appropriate E. coli strains. To ensure the identical conditions for experiments with all plasmids studied, a large batch of the competent cells was prepared for each set of experiments, and the transformations were always done in parallel. The transformants were cultured, harvested, and analyzed under the same conditions. The following E. coli strains were used: AB1157 (37Bachmann B.J. Neidhardt F.D. Derivations and Genotypes of Some Mutant Derivatives of Escherichia coli K-12. American Society for Microbiology, Washington, D. C.1987Google Scholar) as a parent of the recombination deficient strain JC10289 (thr-1, ara-14, leuB6, Δ(gpt-proA)62, lacY1, tsx-33, glnV44(AS), galK2, λ−, rac−, hisG4(Oc), rfbD1, mgl-51, Δ(recA−srl)306, srlR301::Tn10, rpsL31(strR), kdgK51, xylA5, mtl-1, argE3(Oc), thi-1). Strains were obtained from the E. coli Genetic Stock Center, Yale University, New Haven, CT. In the population experiments, the transformation mixture was inoculated into 10-ml LB tubes containing 100 μg/ml ampicillin at a cell density of 102 cells/ml. The cultures were grown at 37 °C with shaking at 250 rpm. At late log phase (A600 ∼1.0 units), the cells were harvested, and the plasmid DNA was isolated as described above and analyzed by restriction digestion. To determine the frequency of recombination, plasmids harboring theGFPuv gene were transformed into the appropriateE. coli strain, plated onto LB plates containing 100 μg/ml ampicillin, and incubated for 16 h at 37 °C. The frequency of recombination was measured as the ratio of the number of white colonies to the total number of viable cells. The white as well as a representative number of fluorescent colonies were inoculated into 10 ml of LB medium (containing ampicillin at 100 μg/ml). After overnight growth, the plasmids were isolated and subjected to the restriction and DNA sequencing analyses. The statistical analyses were performed using SigmaStat version 2.03. This genetic assay enabled the detection and quantitation of the recombination events that occurred directly after transformation of the parental plasmids into the host cells. In order to detect those recombination events that took place at a later stage of colony formation, the recombination product would have to outgrow the parental plasmid molecules (that are present in a large excess at the moment of the recombination event). Consequently, the recombinant plasmid should have a tremendous replication advantage over the parental plasmids. However, this can be easily ruled out by the results of copy number analyses (see "Results"). In addition, the white and the fluorescent colonies are stable. Randomly selected fluorescent colonies (350 total) were inoculated into one bulk culture, mixed, and then plated on plates containing ampicillin. After overnight growth, no white colonies were observed among ∼2 × 105 colonies screened. The same experiment was repeated for the white colonies which revealed no fluorescent colony formation in ∼105 white colonies analyzed. These results indicate that the "color of the colony" (i.e. recombination status of the plasmid) is established at the earliest stage of the colony formation, and masking or overgrowing of the cells to alter the apparent color (e.g. fluorescent cells by the white ones or vice versa) is highly unlikely. To ensure that results of the population experiments are not biased by the growth advantage of cells containing recombination products over the cells containing parental plasmids, the doubling time of E. coli cells harboring either recombination substrates or the recombination products with CTG·CAG tracts of different lengths and orientations was established. The determination of the doubling time and plasmid copy numbers as well as the recombination studies were carried out under identical conditions of bacterial growth (10-ml LB tubes containing 100 μg/ml ampicillin, 37 °C with shaking at 250 rpm). In each case, ∼102–103 cells/ml were used to start the cultures. Aliquots of 10 μl were withdrawn at every 30–60 min for ∼8 h, diluted in LB, and subsequently plated on agar plates without ampicillin. The growth curves were prepared using SigmaPlot 2000 version 6.10, and the doubling time was calculated as described previously (38Bacolla A. Jaworski A. Connors T.D. Wells R.D. J. Biol. Chem. 2001; 276: 18597-18604Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). To exclude the possibility of the replicative advantage of recombination products over the parental plasmids, the copy numbers of these plasmids were determined as described earlier (39Chiang C.S. Bremer H. Plasmid. 1988; 20: 207-220Crossref PubMed Scopus (45) Google Scholar, 40Lin-Chao S. Bremer H. Mol. Gen. Genet. 1986; 203: 143-149Crossref PubMed Scopus (155) Google Scholar); the size of the E. coli genome of 4,639 Kbp (41Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5988) Google Scholar) was used for these calculations. The quantitative analyses of plasmid and genomic DNAs separated by agarose gels were performed using FluorChem version 3.04 (Alpha Innotech Corp.). In order to analyze the products of intramolecular recombination between repeating sequences, the isolated DNAs were linearized with AflIII and labeled by end-filling with the Klenow fragment of E. coli DNA polymerase I and [α-32P]dATP. The labeled DNAs were separated on 1% agarose gels in TAE (40 mm Tris acetate, 1 mmEDTA, pH 8) buffer, and the gels were dried and exposed to x-ray film. The instabilities of the TRS tracts of the recombination products were determined using SphI/BamHI digestion followed by end labeling as described above. The products were resolved in 5–7% polyacrylamide gels in TAE buffer. The lengths of the CTG·CAG inserts were calculated as described earlier (29Pluciennik A. Iyer R.R. Parniewski P. Wells R.D. J. Biol. Chem. 2000; 275: 28386-28397Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The primary structures of more than 35 individual recombination products were determined by direct DNA sequencing of one or both DNA strands. We used an intramolecular plasmid system to study recombination between TRS tracts, where two homologous repetitive sequences are located on the same plasmid molecule and are separated by non-homologous intervening sequences. Two homologous TRS tracts present on the same replicon can be oriented relative to each other as direct or inverted repeats (Fig.1). The term "orientation" is used in this study to define the relative directionality between two recombining homologous sequences (direct and inverted repeats). The terms "orientation I" and "orientation II" refer to the orientation of the TRS sequences relative to the origin of replication; for example, for the plasmids containing (CTG·CAG)n tracts in orientation I, the CTG repeat is in the leading strand template, whereas for the plasmids harboring (CAG·CTG)n tracts, in orientation II, the CTG repeat is in the lagging strand template (28Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. 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Genetics. 2001; 158: 527-540Crossref PubMed Google Scholar). In the case of homologous TRS tracts, the intervening sequence separating the repeats will be also deleted. However, due to their repetitive nature, two homologous CTG·CAG tracts can align and hybridize with each other in several different frames (the number of frames equals the number of repeats divided by 3). As a result of possible different alignments of the CTG·CAG sequences, the length of TRS tracts in the recombination products may vary from the minimum length required for recombination to occur to the maximum length determined by the size of both recombining homologous sequences. The intervening sequence separ
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