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.

Human centrin 2 (HsCen2) is an EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. We studied the structural and Ca(2+)-binding properties of two C-terminal fragments of this protein: SC-HsCen2 (T94-Y172), covering two EF-hands, and LC-HsCen2 (M84-Y172), having 10 additional residues. Both fragments are highly disordered in the apo state but become better structured (although not conformationally homogeneous) in the presence of Ca(2+) and depending on the nature of the cations (K(+) or Na(+)) in the buffer. Only the longer C-terminal domain, in the Ca(2+)-saturated state and in the presence of Na(+) ions, was amenable to structure determination by nuclear magnetic resonance. The solution structure of LC-HsCen2 reveals an open two EF-hand structure, similar to the conformation of related Ca(2+)-saturated regulatory domains. Unexpectedly, the N-terminal helix segment (F86-T94) lies over the exposed hydrophobic cavity. This unusual intramolecular interaction increases considerably the Ca(2+) affinity and constitutes a useful model for the target binding.

The molecular recognition of polyoxometalates by human serum albumin is studied using two different polyoxometalates (POMs) at pH 7.5. The results are compared with those obtained at pH 3.5 and 9.0. At pH 7.5, both POMs strongly interact with the protein with different binding behaviors. The Keggin shaped POM, [H(2)W(12)O(40)](6-) (H2W12), specifically binds the protein, forming a complex with a 1:1 stoichiometry with Ka = 2.9 x 10(6) M(-1). The binding constant decreased dramatically with the increase of the ionic strength, thus indicating a mostly electrostatic binding process. Isothermal titration calorimetry (ITC) experiments show that the binding is an enthalpically driven exothermic process. For the wheel shaped POM [NaP(5)W(30)O(110)](14-) (P5W30), there are up to five binding sites on the protein. Increasing the ionic strength changes the binding behavior significantly, leading to a simple exothermic process, with several binding sites. Competitive binding experiments indicate that the two POMs share one common binding site. In addition, they show the existence of another important binding site for P5W30. The two POMs exhibit different binding dependences on the pH. The combination of the experimental results with the knowledge of the surface map of the protein in its N-B conformation transition domain leads to the proposal for the probable binding site of POMs. The present work reveals a protein conformation change upon P5W30 binding, a new feature not explicitly documented in previous studies.

Human centrin 2 (HsCen2), a member of the EF-hand superfamily of Ca 2؉ -binding proteins, is commonly associated with centrosome-related structures. The protein is organized in two domains, each containing two EFhand motifs, but only the C-terminal half exhibits Ca

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As a step toward the elucidation of the mechanistic pathways governing the known bioactivity of polyoxometalates (POMs), two representative molecules of this class of chemicals, the wheel-shaped [NaP(5)W(30)O(110)]14- (P(5)W(30)) and the Keggin-type anion [H(2)W(12)O(40)]6- (H(2)W(12)), are shown, by two independent techniques, to interact with the fatty-acid-free human serum albumin (HSA). The excited-state lifetime of the single tryptophan molecule of this protein is dramatically decreased by the binding. The quenching mechanism is found to constitute the first example of energy transfer between HSA and POMs. Such molecular recognition is believed to be a key step for subsequent evolution of the systems. Circular dichroism (CD) was used to assess the structural effects of POM binding on HSA and to confirm the interaction revealed by fluorescence studies. CD experiments showed that the two POMs have different effects on the secondary structure of the protein. Binding P(5)W(30) partially unfolds the protein whereas H(2)W(12) has no remarkable effect on the structure of the protein.

Binding human serum albumin (HSA) of three polyoxometalates (POMs) with the Wells-Dawson structure, alpha(2)-[P2W17O61]10- (abbreviated as alpha(2)-P2W17) and two of its metal-substituted derivatives, alpha(2)-[NiP2W17O61]8- and alpha(2)-[CuP2W17O61]8- (alpha(2)-P2W17Ni and alpha(2)-P2W17Cu, respectively) was studied in an aqueous medium at pH 7.5. Fluorescence quenching, circular dichroism (CD), thermal denaturation, and isothermal titration calorimetry (ITC) were used for this purpose. The results were compared with those obtained previously with the Keggin structure POM, [H2W12O40]6- (H2W12), and the wheel-shaped structure, [NaP5W30O110]14- (P5W30). All these POMs bind HSA mainly by electrostatic interactions. Comparison of the physical characteristics and HSA interaction parameters for the POMs of the present work and those studied previously showed that the overall charge of the clusters is not the single parameter governing the binding process and its consequences. In contrast, besides the influences of the structure, the dimension and/or weight of the POMs, the results have permitted highlighting of the importance of each POM atomic composition for its binding behavior.

In Saccharomyces cerevisiae, double-strand breaks (DSBs) activate DNA checkpoint pathways that trigger several responses including a strong G 2 /M arrest. We have previously provided evidence that the phosphatases Ptc2 and Ptc3 of the protein phosphatase 2C type are required for DNA checkpoint inactivation after a DSB and probably dephosphorylate the checkpoint kinase Rad53. In this article we have investigated further the interactions between Ptc2 and Rad53. We showed that forkhead-associated domain 1 (FHA1) of Rad53 interacts with a specific threonine of Ptc2, T376, located outside its catalytic domain in a TXXD motif which constitutes an optimal FHA1 binding sequence in vitro. Mutating T376 abolishes Ptc2 interaction with the Rad53 FHA1 domain and results in adaptation and recovery defects following a DSB. We found that Ckb1 and Ckb2, the regulatory subunits of the protein kinase CK2, are necessary for the in vivo interaction between Ptc2 and the Rad53 FHA1 domain, that Ckb1 binds Ptc2 in vitro and that ckb1⌬ and ckb2⌬ mutants are defective in adaptation and recovery after a DSB. Our data thus strongly suggest that CK2 is the kinase responsible for the in vivo phosphorylation of Ptc2 T376.The DNA checkpoint is a surveillance mechanism that detects DNA lesions or replication blocks and coordinates various responses such as cell cycle arrests and transcriptional or posttranscriptional modifications. This mechanism is present in all eukaryotes and has been particularly analyzed in the yeast Saccharomyces cerevisiae, where it was originally identified (14, 53). In S. cerevisiae, activation of the DNA checkpoint by DNA lesions depends essentially on two sets of proteins, Rad24 and the PCNA-like trimer Rad17-Mec3-Ddc1, on the one hand, and the ATR homolog, the phosphatidylinositol 3-kinase-like Mec1 (in complex with an auxiliary subunit Ddc2), on the other hand (reviewed in references 28 and 58). Both the Rad17-Mec3-Ddc1 and the Mec1-Ddc2 complexes have been shown to be simultaneously and independently recruited to a double-strand break (DSB) artificially induced by the HO endonuclease (15,29). Once activated, Mec1 induces the phosphorylation and the activation of two central transducers, the Rad53 and Chk1 kinases, which subsequently phosphorylate downstream effectors. The phosphorylation of Rad53 and Chk1 also depends on so-called "adaptors," Rad9 in the case of DNA damage and Mrc1 in the case of replication blocks and DNA lesions during S phase (for a review on Rad53 activation, see reference 33).Rad53 plays a central part in S. cerevisiae DNA checkpoint: it controls the majority of the DNA damage responses and rad53⌬ cells are strongly hypersensitive to all genotoxic stresses. Rad53 is the founding member of the conserved family of FHA (forkhead associated) domain-containing checkpoint kinases, which also includes mammalian Chk2 and Schizosaccharomyces pombe Cds1. It contains two FHA domains, FHA1 and FHA2, flanking the protein catalytic domain. FHA domains are protein-protein interaction domains that specifica...