The binding of ethidium bromide to chromatin : model for carcinogen interactions

Five forms of chromatin, which represent the in vivo folded forms of nucleic acid, were isolated and used as the binding substrate for model intercalating compound, ethidium bromide. For all forms of chromatin, the affinity, location and structural effects of binding were examined. On the level of the nucleosome, the binding of ethidium caused a step-wise dissociation of nucleoprotein complex resulting in the initial release of one copy each of H2A and H25 before complete dissociation to free DNA. Dissociation was induced by the intercalation mode of ethidium binding, and was not due to electrostatic interactions or alternate binding modes. The binding of ethidium resulted in no major particle unfolding event prior to step-wise dissociation. Initial ethidium binding to the core particle occurred only at the end 25 by of the core particle DNA, and occurred with very low affinity. Ethidium binding to the 11 nm fiber comprising polynucleosomes, free from H1 and non-histone chromosomal proteins, was characterized by high affinity, very similar to free DNA. The initial binding events were localized within the linker DNA in preference to the folded nucleosomal DNA. In contrast, ethidium binding to the long chromatin form containing H1, the extended 30 nm fiber, displayed a 10-fold lower affinity than either free DNA or the 11 nm fiber. More extensive folding of the 30 nm fiber to its condensed form, induced by the addition of monocations, resulted in a 30-fold decrease in binding affinity of ethidium relative to free DNA. In conclusion, the structure of the chromatin has a large effect on the binding of intercalating compounds. Generally, intercalation into DNA does occur, but the placement of dye molecules appears to be governed by the accessibility of the DNA at the expense of bound histone proteins. The in vitro data were used to construct a model for the interaction of mutagens and carcinogens in vivo and to gain structural information about the native nucleoprotein complexes

Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons

<p>Abstract</p> <p>Background</p> <p>Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion mutation in the coding region of a novel gene. The mechanism of HD is unknown. Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt) contributes to the pathology. However, recent studies have identified early cellular dysfunctions that preclude aggregate formation. Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches. Previously, we demonstrated that tricyclic pyrone (TP) compounds efficiently inhibited formation of amyloid-β (Aβ) aggregates in cell and mouse models representing Alzheimer's Disease (AD). In the present study, we aimed to determine whether TP compounds could prevent aggregation and restore early cellular defects in primary embryonic striatal neurons from animal model representing HD.</p> <p>Results</p> <p>TP compounds effectively inhibit aggregation caused by mhtt in neurons and glial cells. Treatment with TP compounds also alleviated cholesterol accumulation and restored clathrin-independent endocytosis in HD neurons.</p> <p>Conclusion</p> <p>We have found that TP compounds not only blocked mhtt-induced aggregation, but also alleviated early cellular dysfunctions that preclude aggregate formation. Our data suggest TP molecules may be used as lead compounds for prevention or treatment of multiple neurodegenerative diseases including HD and AD.</p

XJB-5-131-mediated improvement in physiology and behaviour of the R6/2 mouse model of Huntington's disease is age- and sex- dependent.

We have reported that the radical scavenger XJB-5-131 attenuates or reverses progression of the disease phenotype in the HdhQ(150/150) mouse, a slow onset model of HD. Here, we tested whether XJB-5-131 has beneficial effects in R6/2 mice, a severe early onset model of HD. We found that XJB-5-131 has beneficial effects in R6/2 mice, by delaying features of the motor and histological phenotype. The impact was sex-dependent, with a stronger effect in male mice. XJB-5-131 treatment improved some locomotor deficits in female R6/2 mice, but the effects were, in general, greater in male mice. Chronic treatment of male R6/2 mice with XJB-5-1-131 reduced weight loss, and improved the motor and temperature regulation deficits, especially in male mice. Treatment with XJB-5-131 had no effect on the lifespan of R6/2 mice. Nevertheless, it significantly slowed somatic expansion at 90 days, and reduced the density of inclusions. Our data show that while treatment with XJB-5-131 had complex effects on the phenotype of R6/2 mice, it produced a number of significant improvements in this severe model of HD

Crosstalk between MSH2–MSH3 and polβ promotes trinucleotide repeat expansion during base excision repair

Studies in knockout mice provide evidence that MSH2–MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2–MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2–MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Nuclease-Deficient FEN-1 Blocks Rad51/BRCA1-Mediated Repair and Causes Trinucleotide Repeat Instability

Previous studies have shown that expansion-prone repeats form structures that inhibit human flap endonuclease (FEN-1). We report here that faulty processing by FEN-1 initiates repeat instability in mammalian cells. Disease-length CAG tracts in Huntington's disease mice heterozygous for FEN-1 display a tendency toward expansions over contractions during intergenerational inheritance compared to those in homozygous wild-type mice. Further, with regard to human cells expressing a nuclease-defective FEN-1, we provide direct evidence that an unprocessed FEN-1 substrate is a precursor to instability. In cells with no endogenous defects in DNA repair, exogenous nuclease-defective FEN-1 causes repeat instability and aberrant DNA repair. Inefficient flap processing blocks the formation of Rad51/BRCA1 complexes but invokes repair by other pathways