Opposing effects on the cell cycle of T lymphocytes by Fbxo7 via Cdk6 and p27

G₁ phase cell cycle proteins, such as cyclin-dependent kinase 6 (Cdk6) and its activating partners, the D-type cyclins, are important regulators of T-cell development and function. An F-box protein, called F-box only protein 7 (Fbxo7), acts as a cell cycle regulator by enhancing cyclin D-Cdk6 complex formation and stabilising levels of p27, a cyclin-dependent kinase inhibitor. We generated a murine model of reduced Fbxo7 expression to test its physiological role in multiple tissues and found that these mice displayed a pronounced thymic hypoplasia. Further analysis revealed that Fbxo7 differentially affected proliferation and apoptosis of thymocytes at various stages of differentiation in the thymus and also mature T-cell function and proliferation in the periphery. Paradoxically, Fbxo7-deficient immature thymocytes failed to undergo expansion in the thymus due to a lack of Cdk6 activity, while mature T cells showed enhanced proliferative capacity upon T-cell receptor engagement due to reduced p27 levels. Our studies reveal differential cell cycle regulation by Fbxo7 at different stages in T-cell development.     

PTC124 targets genetic disorders caused by nonsense mutations

Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.     

Coupled 142Nd-143Nd evidence for a protracted magma ocean in Mars

Resolving early silicate differentiation timescales is crucial for understanding the chemical evolution and thermal histories of terrestrial planets. Planetary-scale magma oceans are thought to have formed during early stages of differentiation, but the longevity of such magma oceans is poorly constrained. In Mars, the absence of vigorous convection and plate tectonics has limited the scale of compositional mixing within its interior, thus preserving the early stages of planetary differentiation. The SNC (Shergotty-Nakhla-Chassigny) meteorites from Mars retain 'memory' of these events. Here we apply the short-lived 146Sm-142Nd and the long-lived 147Sm-143Nd chronometers to a suite of shergottites to unravel the history of early silicate differentiation in Mars. Our data are best explained by progressive crystallization of a magma ocean with a duration of approximately 100 million years after core formation. This prolonged solidification requires the existence of a primitive thick atmosphere on Mars that reduces the cooling rate of the interior.     

The structural basis of yeast prion strain variants

Among the many surprises to arise from studies of prion biology, perhaps the most unexpected is the strain phenomenon whereby a single protein can misfold into structurally distinct, infectious states that cause distinguishable phenotypes. Similarly, proteins can adopt a spectrum of conformations in non-infectious diseases of protein folding; some are toxic and others are well tolerated. However, our understanding of the structural differences underlying prion strains and how these differences alter their physiological impact remains limited. Here we use a combination of solution NMR, amide hydrogen/deuterium (H/D) exchange and mutagenesis to study the structural differences between two strain conformations, termed Sc4 and Sc37 (ref. 5), of the yeast Sup35 prion. We find that these two strains have an overlapping amyloid core spanning most of the Gln/Asn-rich first 40 amino acids that is highly protected from H/D exchange and very sensitive to mutation. These features indicate that the cores are composed of tightly packed beta-sheets possibly resembling 'steric zipper' structures revealed by X-ray crystallography of Sup35-derived peptides. The stable structure is greatly expanded in the Sc37 conformation to encompass the first 70 amino acids, revealing why this strain shows increased fibre stability and decreased ability to undergo chaperone-mediated replication. Our findings establish that prion strains involve large-scale conformational differences and provide a structural basis for understanding a broad range of functional studies, including how conformational changes alter the physiological impact of prion strains.     

ATP-induced helicase slippage reveals highly coordinated subunits

Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. Bacteriophage T7 gene product 4 is a model hexameric helicase that has been observed to use dTTP, but not ATP, to unwind double-stranded (ds)DNA as it translocates from 5' to 3' along single-stranded (ss)DNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found that T7 helicase binds and hydrolyses ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective maximum rates V(max), Michaelis constants K(M) and concentrations. In contrast, processivity does not follow a simple competitive behaviour and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus they interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.     

Maize HapMap2 identifies extant variation from a genome in flux

Whereas breeders have exploited diversity in maize for yield improvements, there has been limited progress in using beneficial alleles in undomesticated varieties. Characterizing standing variation in this complex genome has been challenging, with only a small fraction of it described to date. Using a population genetics scoring model, we identified 55 million SNPs in 103 lines across pre-domestication and domesticated Zea mays varieties, including a representative from the sister genus Tripsacum. We find that structural variations are pervasive in the Z. mays genome and are enriched at loci associated with important traits. By investigating the drivers of genome size variation, we find that the larger Tripsacum genome can be explained by transposable element abundance rather than an allopolyploid origin. In contrast, intraspecies genome size variation seems to be controlled by chromosomal knob content. There is tremendous overlap in key gene content in maize and Tripsacum, suggesting that adaptations from Tripsacum (for example, perennialism and frost and drought tolerance) can likely be integrated into maize.     

Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia

The evolutionarily conserved SLX4 protein, a key regulator of nucleases, is critical for DNA damage response. SLX4 nuclease complexes mediate repair during replication and can also resolve Holliday junctions formed during homologous recombination. Here we describe the phenotype of the Btbd12 knockout mouse, the mouse ortholog of SLX4, which recapitulates many key features of the human genetic illness Fanconi anemia. Btbd12-deficient animals are born at sub-Mendelian ratios, have greatly reduced fertility, are developmentally compromised and are prone to blood cytopenias. Btbd12(-/-) cells prematurely senesce, spontaneously accumulate damaged chromosomes and are particularly sensitive to DNA crosslinking agents. Genetic complementation reveals a crucial requirement for Btbd12 (also known as Slx4) to interact with the structure-specific endonuclease Xpf-Ercc1 to promote crosslink repair. The Btbd12 knockout mouse therefore establishes a disease model for Fanconi anemia and genetically links a regulator of nuclease incision complexes to the Fanconi anemia DNA crosslink repair pathway.