The human tissue nonspecific alkaline phosphatase (TNAP) is found in liver, kidney, and bone. Mutations in the TNAP gene can lead to Hypophosphatasia, a rare inborn disease that is characterized by defective bone mineralization. TNAP is 74% homologous to human placental alkaline phosphatase (PLAP) whose crystal structure has been recently determined at atomic resolution (Le Du, M. H., Stigbrand, T., Taussig The alkaline phosphatases (EC 3.1.3.1) (APs) 1 form a large family of dimeric enzymes common to all organisms. They catalyze the hydrolysis of phosphomonoesters (1) with release of inorganic phosphate. Mammalian APs have low sequence identity with the Escherichia coli enzyme (25-30%), although the residues involved in the active site of the enzyme and the ligands coordinating the two zinc atoms and the magnesium ion are largely conserved. Therefore, the catalytic mechanism deduced from the structure of the E. coli AP is believed to be similar in eukaryotic APs (2). This mechanism involves the activation of the catalytic serine by a zinc atom, the formation of a covalent phosphoseryl intermediate, the hydrolysis of the phosphoseryl by a water molecule activated by a second zinc atom, and the release of the phosphate product or its transfer to a phosphate acceptor (3).In humans, three out of four AP isozymes are tissue-specific: one is placental (PLAP), the second appears in germ cells (GCAP), and the third in the intestine (IAP). They are 90 -98% homologous, and their genes are clustered on chromosome 2q37.1. The fourth, TNAP, 50% identical to the other three, is nonspecific and can be found in bone, liver, and kidney (4, 5, 6). Its gene is located on chromosome 1p34 -36 (7), and mutations in the TNAP gene have been associated with hypophosphatasia, a rare inherited disorder, characterized by defective bone mineralization. The disease is highly variable in its clinical expression, due to the strong allelic heterogeneity in the TNAP gene. Such expression ranges from stillbirth without mineralized bone to pathological fractures developing only late in adulthood (8). Depending on the age of onset, five clinical forms are currently recognized: perinatal, infantile, childhood, adult, and odontohypophosphatasia. To date, 89 different mutations associated with this disease have been characterized (9 -22). Correlation between genotype and phenotype are difficult to establish, because most patients are compound heterozygous for missense mutations, making difficult the determination of the respective roles of each mutation.This difficulty arises mainly from the lack of data concerning the precise role that TNAP plays in bone mineralization. This may be partly solved by the use of site-directed mutagenesis of TNAP cDNA and cell transfection to assay residual activity of the mutant AP enzyme (16,18,20,(23)(24)(25). However, this only measures the ability of the enzyme to hydrolyze phosphomonoesters. Transfection assays cannot distinguish structural mutations from functional ones, and mutations that exhibit residual activi...

SUMMARY The shelterin protein protects telomeres against activation of the DNA damage checkpoint and recombinational repair. We show here that a dimer of the shelterin subunit TRF2 wraps ~90 bp of DNA through several lysine and arginine residues localized around its homodimerization domain. The expression of a wrapping-deficient TRF2 mutant, named Top-less, alters telomeric DNA topology, decreases the number of terminal loops (t-loops), and triggers the ATM checkpoint, while still protecting telomeres against non-homologous end joining (NHEJ). In Top-less cells, the protection against NHEJ is alleviated if the expression of the TRF2-interacting protein RAP1 is reduced. We conclude that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ. The presence of RAP1 at telomeres appears as a backup mechanism to prevent NHEJ when topology-mediated telomere protection is impaired.