Keiichi Ohshima

Inhibitory effects of expanded GAA.TTC triplet repeats from intron I of the Friedreich ataxia gene on transcription and replication in vivo.

Friedreich ataxia (FRDA) is associated with the expansion of a GAA·TTC triplet repeat in the first intron of the frataxin gene, resulting in reduced levels of frataxin mRNA and protein. To investigate the mechanisms by which the intronic expansion produces its effect, GAA·TTC repeats of various lengths (9 to 270 triplets) were cloned in both orientations in the intron of a reporter gene. Plasmids containing these repeats were transiently transfected into COS-7 cells. A length- and orientation-dependent inhibition of reporter gene expression was observed. RNase protection and Northern blot analyses showed very low levels of mature mRNA when longer GAA repeats were transcribed, with no accumulation of primary transcript. Replication of plasmids carrying long GAA·TTC tracts (∼250 triplets) was greatly inhibited in COS-7 cells compared with plasmids carrying (GAA·TTC)9 and (GAA·TTC)90. Replication inhibition was five times greater for the plasmid whose transcript contains (GAA)230than for the plasmid whose transcript contains (UUC)270. Our in vivo investigation revealed that expanded GAA·TTC repeats from intron I of the FRDA gene inhibit transcription rather than post-transcriptional RNA processing and also interfere with replication. The molecular basis for these effects may be the formation of non-B DNA structures.

Sticky DNA:Self-Association Properties of Long GAA·TTC Repeats in R·R·Y Triplex Structures from Friedreich’s Ataxia

A novel DNA structure, sticky DNA, is described for lengths of (GAA.TTC)n found in intron 1 of the frataxin gene of Friedreichs ataxia patients. Sticky DNA is formed by the association of two purine.purine.pyrimidine (R.R.Y) triplexes in negatively supercoiled plasmids at neutral pH. An excellent correlation was found between the lengths of (GAA.TTC) (> 59 repeats): first, in FRDA patients, second, required to inhibit transcription in vivo and in vitro, and third, required to adopt the sticky conformation. Fourth, (GAAGGA.TCCTTC)65, also found in intron 1, does not form sticky DNA, inhibit transcription, or associate with the disease. Hence, R.R.Y triplexes and/or sticky DNA may be involved in the etiology of FRDA.

Pausing of DNA Synthesis in Vitro at Specific Loci in CTG and CGG Triplet Repeats from Human Hereditary Disease Genes

Several human hereditary neuromuscular disease genes are associated with the expansion of CTG or CGG triplet repeats. The DNA syntheses of CTG triplets ranging from 17 to 180 and CGG repeats from 9 to 160 repeats in length were studied in vitro. Primer extensions using the Klenow fragment of DNA polymerase I, the modified T7 DNA polymerase (Sequenase), or the human DNA polymerase β paused strongly at specific loci in the CTG repeats. The pausings were abolished by heating at 70°C. As the length of the triplet repeats in duplex DNA, but not in single-stranded DNA, was increased, the magnitude of pausings increased. The location of the pause sites was determined by the distance between the site of primer hybridization and the beginning of the triplet repeats. CGG triplet repeats also showed similar, but not identical, patterns of pausings. These results indicate that appropriate lengths of the triplets adopt a non-B conformation(s) that blocks DNA polymerase progression; the resultant idling polymerase may catalyze slippages to give expanded sequences and hence provide the molecular basis for this non-Mendelian genetic process. These mechanisms, if present in human cells, may be related to the etiology of certain neuromuscular diseases such as myotonic dystrophy and Fragile X syndrome.

Frataxin knockin mouse

Friedreich ataxia is the consequence of frataxin deficiency, most often caused by a GAA repeat expansion in intron 1 of the corresponding gene. Frataxin is a mitochondrial protein involved in iron homeostasis. As an attempt to generate a mouse model of the disease, we introduced a (GAA)230 repeat within the mouse frataxin gene by homologous recombination. GAA repeat knockin mice were crossed with frataxin knockout mice to obtain double heterozygous mice expressing 25–36% of wild‐type frataxin levels. These mice were viable and did not develop anomalies of motor coordination, iron metabolism or response to iron loading. Repeats were meiotically and mitotically stable.

Cloning, characterization, and properties of seven triplet repeat DNA sequences.

Several neuromuscular and neurodegenerative diseases are caused by genetically unstable triplet repeat sequences (CTG·CAG, CGG·CCG, or AAG·CTT) in or near the responsible genes. We implemented novel cloning strategies with chemically synthesized oligonucleotides to clone seven of the triplet repeat sequences (GTA·TAC, GAT·ATC, GTT·AAC, CAC·GTG, AGG·CCT, TCG·CGA, and AAG·CTT), and the adjoining paper (Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D. (1996) J. Biol. Chem. 271, 16784-16791) describes studies on TTA·TAA. This approach in conjunction with in vivo expansion studies in Escherichia coli enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of ∼16 up to 158 triplets in both orientations with varying extents of polymorphisms. The inserts were characterized by DNA sequencing as well as DNA polymerase pausings, two-dimensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA conformations. AAG·CTT and AGG·CCT form intramolecular triplexes, and the other five repeat sequences do not form any previously characterized non-B structures. However, long tracts of TCG·CGA showed strong inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG·CAG and CGG·CCG (Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., and Wells, R. D. (1995) J. Biol. Chem. 270, 27014-27021). This work along with other studies (Wells, R. D. (1996) J. Biol. Chem. 271, 2875-2878) on CTG·CAG, CGG·CCG, and TTA·TAA makes available long inserts of all 10 triplet repeat sequences for a variety of physical, molecular biological, genetic, and medical investigations. A model to explain the reduction in mRNA abundance in Friedreichs ataxia based on intermolecular triplex formation is proposed.

FLEXIBLE DNA : GENETICALLY UNSTABLE CTG.CAG AND CGG.CCG FROM HUMAN HEREDITARY NEUROMUSCULAR DISEASE GENES

The properties of duplex CTG·CAG and CGG·CCG, which are involved in the etiology of several hereditary neurodegenerative diseases, were investigated by a variety of methods, including circularization kinetics, apparent helical repeat determination, and polyacrylamide gel electrophoresis. The bending moduli were 1.13 × 10−19 erg·cm for CTG and 1.27 × 10−19 erg·cm for CGG, ∼40% less than for random B-DNA. Also, the persistence lengths of the triplet repeat sequences were ∼60% the value for random B-DNA. However, the torsional moduli and the helical repeats were 2.3 × 10−19 erg·cm and 10.4 base pairs (bp)/turn for CTG and 2.4 × 10−19 erg·cm and 10.3 bp/turn for CGG, respectively, all within the range for random B-DNA. Determination of the apparent helical repeat by the band shift assay indicated that the writhe of the repeats was different from that of random B-DNA. In addition, molecules of 224–245 bp in length (64–71 triplet repeats) were able to form topological isomers upon cyclization. The low bending moduli are consistent with predictions from crystallographic variations in slide, roll, and tilt. No unpaired bases or non-B-DNA structures could be detected by chemical and enzymatic probe analyses, two-dimensional agarose gel electrophoresis, and immunological studies. Hence, CTG and CGG are more flexible and highly writhed than random B-DNA and thus would be expected to act as sinks for the accumulation of superhelical density.

Hairpin formation during DNA synthesis primer realignment in vitro in triplet repeat sequences from human hereditary disease genes.

Genetic expansion of DNA triplet repeat sequences (TRS) found in neurogenetic disorders may be due to abnormal DNA replication. We have previously observed strong DNA synthesis pausings at specific loci within the long tracts (>∼70 repeats) of CTG·CAG and CGG·CCG as well as GTC·GAC by primer extensions in vitro using DNA polymerases (the Klenow fragment ofEscherichia coli DNA polymerase I, the modified T7 DNA polymerase (Sequenase), and human DNA polymerase β). Herein, we have isolated and analyzed the products of stalled synthesis found at ∼30–40 triplets from the beginning of the TRS. DNA sequence analyses revealed that the stalled products contained short tracts of homogeneous TRS (6–12 repeats) in the middle of the sequence corresponding to the flanking region of the primer-template sequence. The sequence at the 3′-side terminated at the end of the primer, indicating that the primer molecule had served as a template. In addition, chemical probe and polyacrylamide gel electrophoretic analyses revealed that the stalled products existed in hairpin structures. We postulate that these products of the DNA polymerases are caused by the existence of an unusual DNA conformation(s) within the TRS, during the in vitro DNA synthesis, enhancing the DNA slippages and the hairpin formations in the TRS due to primer realignment. The consequence of these steps is DNA synthesis to the end of the primer and termination. Primer realignment including hairpin formation may play an important intermediate role in the replication of TRS in vivo to elicit genetic expansions.

Knock-out of the cyaY gene in Escherichia coli does not affect cellular iron content and sensitivity to oxidants.

Friedreich ataxia is a recessively inherited neurodegenerative disease caused by deficiency of a highly conserved mitochondrial protein, frataxin. Frataxin deficiency results in mitochondrial iron accumulation and oxidative stress. Frataxin shows homology with the CyaY proteins of γ‐purple bacteria, whose function is unknown. We knocked out the CyaY gene in Escherichia coli MM383 by homologous recombination and we generated an E. coli MM383 strain overexpressing CyaY. Bacterial growth, iron content and survival after exposure to H2O2 did not differ among these strains, suggesting that, despite structural similarities, cyaY proteins in bacteria may have a different function from frataxin homologues in mitochondria.

PACAP protects neuronal PC12 cells from the cytotoxicity of human prion protein fragment 106–126

Misfolding of the prion protein yields amyloidogenic isoforms, and it shows exacerbating neuronal damage in neurodegenerative disorders including prion diseases. Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) potently stimulate neuritogenesis and survival of neuronal cells in the central nervous system. Here, we tested these neuropeptides on neurotoxicity in PC12 cells induced by the prion protein fragment 106–126 [PrP (106–126)]. Concomitant application of neuropeptide with PrP(106–126) (5×10−5 M) inhibited the delayed death of neuron‐like PC12 cells. In particular, PACAP27 inhibited the neurotoxicity of PrP(106–126) at low concentrations (>10−15 M), characterized by the deactivation of PrP(106–126)‐stimulated caspase‐3. The neuroprotective effect of PACAP27 was antagonized by the selective PKA inhibitor, H89, or the MAP kinase inhibitor, U0126. These results suggest that PACAP27 attenuates PrP(106–126)‐induced delayed neurotoxicity in PC12 cells by activating both PKA and MAP kinases mediated by PAC1 receptor.

A nonpathogenic GAAGGA repeat in the Friedreich gene: Implications for pathogenesis

Article abstract An individual with late-onset ataxia was found to be heterozygous for an unusual (GAAGGA)65 sequence and a normal GAA repeat in the frataxin gene. No frataxin point mutation was present, excluding a form of Friedreich ataxia. (GAAGGA)65 did not have the inhibitory effect on gene expression in transfected cells shown by pathogenic GAA repeats of similar length. GAA repeats, but not (GAAGGA)65, adopt a triple helical conformation in vitro. We suggest that such a triplex structure is essential for suppression of gene expression.