The mechanism by which trinucleotide expansion occurs in human genes is not understood. However, it has been hypothesized that DNA secondary structure may actively participate by preventing FEN-1 cleavage of displaced Okazaki fragments. We show here that secondary structure can, indeed, play a role in expansion by a FEN-1-dependent mechanism. Secondary structure inhibits flap processing at CAG, CGG, or CTG repeats in a length-dependent manner by concealing the 5' end of the flap that is necessary for both binding and cleavage by FEN-1. Thus, secondary structure can defeat the protective function of FEN-1, leading to site-specific expansions. However, when FEN-1 is absent from the cell, alternative pathways to simple inhibition of flap processing contribute to expansion.

Inordinate expansion and hypermethylation of the fragile X DNA triplet repeat, (GGC).-(GCC)., are correlated with the ability of the individual G-and C-rich single strands to form hairpin structures. Two-dimensional NMR and gel electrophoresis studies show that both the Gand C-rich single strands form hairpins under physiological conditions. This propensity of hairpin formation is more pronounced for the C-rich strand than for the G-rich strand. This observation suggests that the C-rich strand is more likely to form hairpin or "slippage" structure and show asymmetric strand expansion during replication. NMR data also show that the hairpins formed by the C-rich strands fold in such a way that the cytosine at the CpG step of the stem is C-C paired.The presence ofa C C mismatch at the CpG site generates local flexibility, thereby providing analogs of the transition to the methyltransferase. In other words, the hairpins of the C-rich strand act as better substrates for the human methyltransferase than the Watson-Crick duplex or the G-rich strand. Therefore, hairpin formation could account for the specific methylation of the CpG island in the fragile X repeat that occurs during inactivation of the FMR1 gene during the onset of the disease.Simple tandemly repeated DNA sequences are interspersed in both transcribed and nontranscribed regions of chromosomes (1-3). The hypothesis (4) that the unusual DNA structures adopted by these repeats principally determine their specific functions is gaining strength. We have previously described the unusual hairpin structures (5, 6) adopted by a variety of repetitive DNA sequences. Here, we show by NMR and gel electrophoresis that the individual strands from the fragile X triplet repeats, (GGC)n-(GCC)n, form intramolecular hairpins under physiological conditions. In these hairpins, the number of Watson-Crick G-C pairs is maximized in the stem through the formation of G-G or C C mispairs flanked by G-C pairs (Fig. 1). As shown below, these hairpins provide structural basis for three major phenomena associated with the fragile X syndrome (3, 4): (i) the site-specific fragility, (ii) the amplification of the repeat (especially the preferential expansion of the C-rich strand), and (iii) the hypermethylation of the CpG island adjacent to the fragile X gene, FMRI. MATERIALS AND METHODSGel Electrophoresis. Oligonucleotides were fully denatured by heating at 95°C for 2 min in 5 mM Tris/1 mM EDTA buffer, pH 7.5, containing 5 mM or 200 mM NaCl, followed by incubation at 55°C for 10 min and gradual cooling to roomThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Note that in the hairpins of the G-and C-rich strands, the central mismatched G-G or C-C pair in the stem is surrounded by twoWatson-Crick G-C pairs. IITo whom reprint requests should be addressed. 5199

The oxidation and toxicity of dopamine is believed to contribute to the selective neurodegeneration associated with Parkinson disease. The formation of reactive radicals and quinones greatly contributes to dopaminergic toxicity through a variety of mechanisms. The physiological metabolism of dopamine to 3,4-dihydroxyphenylacetaldehyde (DOPAL) via monoamine oxidase significantly increases its toxicity. To more adequately explain this enhanced toxicity, we hypothesized that DOPAL is capable of forming radical and quinone species upon oxidation. Here, two unique oxidation products of DOPAL are identified. Several different oxidation methods gave rise to a transient DOPAL semiquinone radical, which was characterized by electron paramagnetic resonance spectroscopy. NMR identified the second oxidation product of DOPAL as the ortho-quinone. Also, carbonyl hydration of DOPAL in aqueous media was evident via NMR. Interestingly, the DOPAL quinone exists exclusively in the hydrated form. Furthermore, the enzymatic and chemical oxidation of DOPAL greatly enhance protein cross-linking, whereas auto-oxidation results in the production of superoxide. Also, DOPAL was shown to be susceptible to oxidation by cyclooxygenase-2 (COX-2). The involvement of this physiologically relevant enzyme in both oxidative stress and Parkinson disease underscores the potential importance of DOPAL in the pathogenesis of this condition. Parkinson disease (PD)2 involves specific loss of dopaminergic nuclei in the substantia nigra of the brain (1). Although the exact causes of this selective degeneration are unknown (2), the role of affected cells as centers of dopamine (DA) synthesis, storage, and metabolism suggests that DA may be an endogenous neurotoxin (3, 4) that contributes to the pathogenesis of PD. DA may act as a source of cellular oxidative stress and is known to undergo oxidation (Scheme 1) to cytotoxic radicals and quinones (5-8). Such oxidations can occur spontaneously or via metal-or enzyme-catalyzed mechanisms (9). One-electron oxidation of DA produces a radical capable of interfering with DA storage and causing oxidative protein and DNA modifications (5, 6, 10). Similarly, two-electron oxidation of DA to an ortho-quinone results in reactivity with cellular nucleophiles such as thiols and proteins (8, 11). Both species are capable of redox cycling, which could deplete cellular oxidative defenses. Also, in the presence of transition metals and/or O 2 , such oxidations could result in the production of ROS capable of inducing lipid peroxidation and damage to other cellular macromolecules (12). Another potential mechanism of toxicity for DA is its physiological metabolism to 3,4-dihydroxyphenylacetaldehyde (DOPAL) (13).DOPAL is a very reactive aldehyde that is 100 -1000-fold more toxic than DA both in vivo and in vitro (14,15). Physiological levels of DOPAL in the substantia nigra are ϳ2 M; levels as low as 6 M can exert significant toxicity (16). Reactivity with proteins, presumably via Schiff base formation, is an important mechanism o...

Human mucins are T or S glycosylated tandem repeat proteins. In breast cancer, mucins become under or unglycosylated. Two-dimensional nuclear magnetic resonance experiments are performed on chemically synthesized mucin tandem repeat polypeptides, (PDTRPAPGST-APPAHGVTSA)n the unglycosylated form for n=1,3 where (APDTR) constitutes the antigenic sites for the antibodies isolated form the tumors in the breast cancer patients. These studies demonstrate how the tandem repeats assemble in space giving rise to the overall tertiary structure, and the local structure and presentation of the antigenic site(APDTR) at the junction of two neighboring repeats. The NMR data reveal repeating knob-like structures connected by extended spacers. The knobs protrude away from the long-axis of Muc-1 and the predominant antigenic site (APDTR) forms the accessible tip of the knob. Multiple tandem repeats enhance the rigidity and presentation of the knob-like structures.

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