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Failure of cell cleavage induces senescence in tetraploid primary cells

Mol Biol Cell. 2014; pii: mbc.E14-03-0844.

Panopoulos A, Pacios-Bras C, Choi J, Yenjerla M, Sussman MA, Fotedar R, Margolis RL.

Mol Biol Cell. 2014; pii: mbc.E14-03-0844Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control.

We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts (HFF) become senescent in tetraploid G1 following drug or siRNA induced failure of cell cleavage. In contrast, T-antigen transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle following cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker, and of the FUCCI late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid.

We conclude that tetraploid primary cells can become senescent without DNA damage, and that induction of senescence is critical to tetraploidy arrest.

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated genetic recombination

J Biol Chem. 2014; pii: jbc.M114.577924.

Yadav T, Carrasco B, Serrano E, Alonso JC.

J Biol Chem. 2014; pii: jbc.M114.577924Bacillus subtilis competence-induced RecA, SsbA, SsbB and DprA are required to internalize and to recombine single-stranded (ss) DNA with homologous resident duplex. RecA, in the ATP-Mg2+ bound form (RecA-ATP), can nucleate and form filament onto ssDNA, but inactive to catalyze DNA recombination.

We report that SsbA or SsbB bound to ssDNA blocks the RecA filament formation, and fail to activate recombination. DprA facilitates RecA filamentation, however the filaments cannot engage in DNA recombination. When ssDNA was pre-incubated with SsbA, but not SsbB, DprA was able to activate DNA strand exchange dependent on RecA-ATP. This work demonstrates that RecA-ATP, in concert with SsbA and DprA, catalyzes DNA strand exchange, and SsbB is an accessory factor in the reaction. In contrast, RecA-dATP efficiently catalyzes strand exchange even in the absence of SSBs or DprA, and addition of the accessory factors marginally improved it.

We proposed that the RecA bound nucleotide (ATP and to a lesser extent dATP) might dictate the requirement for accessory factors.

Direct measurement of the dielectric polarization properties of DNA

Proc Natl Acad Sci USA. 2014; pii: 201405702.

Cuervo A, Dans PD, Carrascosa JL, Orozco M, Gomila G, Fumagalli L.

Proc Natl Acad Sci USA. 2014; pii: 201405702The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it.

Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ∼ 2–4), we found that the DNA dielectric constant is ∼8, considerably higher than the value of ∼3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson–Boltzmann methods.

Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques.

Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure

Nucleic Acids Res. 2014; 42 (11): 7421-7428.

Suzuki Y, Endo M, Cañas C, Ayora S, Alonso JC, Sugiyama H, Takeyasu K.

Nucleic Acids Res. 2014; 42 (11): 7421-7428Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction.

Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme.

This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.



Crystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage K

Virol J. 2014; 11 (1) :133.

Sanz-Gaitero M, Keary R, Garcia-Doval C, Coffey A, van Raaij MJ.

Virol J. 2014; 11 (1) :133Background: Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge.

Methods: The CHAPK domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants.

Results: The structure of CHAPK revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAPK contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity.

Conclusions: The high-resolution structure of the CHAPK domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAPK with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity.