Institute Biologia Funcional Y Genomica CSIC

Salamanca, Spain

Institute Biologia Funcional Y Genomica CSIC

Salamanca, Spain
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PubMed | Institute Biologia Funcional y Genomica CSIC, Cornell University and CSIC - National Center for Biotechnology
Type: Journal Article | Journal: Nucleic acids research | Year: 2015

The Ku heterodimer serves in the initial step in repairing DNA double-strand breaks by the non-homologous end-joining pathway. Besides this key function, Ku also plays a role in other cellular processes including telomere maintenance. Inactivation of Ku can lead to DNA repair defects and telomere aberrations. In model organisms where Ku has been studied, inactivation can lead to DNA repair defects and telomere aberrations. In general Ku deficient mutants are viable, but a notable exception to this is human where Ku has been found to be essential. Here we report that similar to the situation in human Ku is required for cell proliferation in the fungus Ustilago maydis. Using conditional strains for Ku expression, we found that cells arrest permanently in G2 phase when Ku expression is turned off. Arrest results from cell cycle checkpoint activation due to persistent signaling via the DNA damage response (DDR). Our results point to the telomeres as the most likely source of the DNA damage signal. Inactivation of the DDR makes the Ku complex dispensable for proliferation in this organism. Our findings suggest that in U. maydis, unprotected telomeres arising from Ku depletion are the source of the signal that activates the DDR leading to cell cycle arrest.


Yu E.Y.,Cornell University | Perez-Martin J.,Institute Biologia Funcional y Genomica CSIC | Holloman W.K.,Cornell University | Holloman W.K.,New York Medical College | And 2 more authors.
PLoS Genetics | Year: 2015

A subset of human cancer cells uses a specialized, aberrant recombination pathway known as ALT to maintain telomeres, which in these cells are characterized by complex aberrations including length heterogeneity, high levels of unpaired C-strand, and accumulation of extra-chromosomal telomere repeats (ECTR). These phenotypes have not been recapitulated in any standard budding or fission yeast mutant. We found that eliminating Ku70 or Ku80 in the yeast-like fungus Ustilago maydis results initially in all the characteristic telomere aberrations of ALT cancer cells, including C-circles, a highly specific marker of ALT. Subsequently the ku mutants experience permanent G2 cell cycle arrest, accompanied by loss of telomere repeats from chromosome ends and even more drastic accumulation of very short ECTRs (vsECTRs). The deletion of atr1 or chk1 rescued the lethality of the ku mutant, and “trapped” the telomere aberrations in the early ALT-like stage. Telomere abnormalities are telomerase-independent, but dramatically suppressed by deletion of mre11 or blm, suggesting major roles for these factors in the induction of the ALT pathway. In contrast, removal of other DNA damage response and repair factors such as Rad51 has disparate effects on the ALT phenotypes, suggesting that these factors process ALT intermediates or products. Notably, the antagonism of Ku and Mre11 in the induction of ALT is reminiscent of their roles in DSB resection, in which Blm is also known to play a key role. We suggest that an aberrant resection reaction may constitute an early trigger for ALT telomeres, and that the outcomes of ALT are distinct from DSB because of the unique telomere nucleoprotein structure. © 2015 Yu et al.


PubMed | Institute Biologia Funcional y Genomica CSIC, Cornell University and New York Medical College
Type: Journal Article | Journal: PLoS genetics | Year: 2015

A subset of human cancer cells uses a specialized, aberrant recombination pathway known as ALT to maintain telomeres, which in these cells are characterized by complex aberrations including length heterogeneity, high levels of unpaired C-strand, and accumulation of extra-chromosomal telomere repeats (ECTR). These phenotypes have not been recapitulated in any standard budding or fission yeast mutant. We found that eliminating Ku70 or Ku80 in the yeast-like fungus Ustilago maydis results initially in all the characteristic telomere aberrations of ALT cancer cells, including C-circles, a highly specific marker of ALT. Subsequently the ku mutants experience permanent G2 cell cycle arrest, accompanied by loss of telomere repeats from chromosome ends and even more drastic accumulation of very short ECTRs (vsECTRs). The deletion of atr1 or chk1 rescued the lethality of the ku mutant, and trapped the telomere aberrations in the early ALT-like stage. Telomere abnormalities are telomerase-independent, but dramatically suppressed by deletion of mre11 or blm, suggesting major roles for these factors in the induction of the ALT pathway. In contrast, removal of other DNA damage response and repair factors such as Rad51 has disparate effects on the ALT phenotypes, suggesting that these factors process ALT intermediates or products. Notably, the antagonism of Ku and Mre11 in the induction of ALT is reminiscent of their roles in DSB resection, in which Blm is also known to play a key role. We suggest that an aberrant resection reaction may constitute an early trigger for ALT telomeres, and that the outcomes of ALT are distinct from DSB because of the unique telomere nucleoprotein structure.


Sartore E.,Institute Biologia Funcional y Genomica CSIC | Sartore E.,University of California at Berkeley | Perez-Martin J.,Institute Biologia Funcional y Genomica CSIC
Journal of Cell Science | Year: 2012

The morphogenesis-related NDR kinase (MOR) pathway regulates morphogenesis in fungi. In spite of the high conservation of its components, impairing their functions results in highly divergent cellular responses depending on the fungal species. The reasons for such differences are unclear. Here we propose that the species-specific connections between cell cycle regulation and the MOR pathway could be partly responsible for these divergences. We based our conclusion on the characterization of the MOR pathway in the fungus Ustilago maydis. Each gene that encodes proteins of this pathway in U. maydis was deleted. All mutants exhibited a constitutive hyperpolarized growth, contrasting with the loss of polarity observed in other fungi. Using a conditional allele of the central NDR kinase Ukc1, we found that impairing MOR function resulted in a prolonged G2 phase. This cell cycle delay appears to be the consequence of an increase in Cdk1 inhibitory phosphorylation. Strikingly, prevention of the inhibitory Cdk1 phosphorylation abolished the hyperpolarized growth associated with MOR pathway depletion. We found that the prolonged G2 phase resulted in higher levels of expression of crk1, a conserved kinase that promotes polar growth in U. maydis. Deletion of crk1 also abolished the dramatic activation of polar growth in cells lacking the MOR pathway. Taken together, our results suggest that Cdk1 inhibitory phosphorylation may act as an integrator of signaling cascades regulating fungal morphogenesis and that the distinct morphological response observed in U. maydis upon impairment of the MOR pathway could be due to a cell cycle deregulation. © 2012.


Tenorio-Gomez M.,Institute Biologia Funcional Y Genomica CSIC | De Sena-Tomas C.,Institute Biologia Funcional Y Genomica CSIC | Perez-Martin J.,Institute Biologia Funcional Y Genomica CSIC
PLoS ONE | Year: 2015

DNA damage response (DDR) leads to DNA repair, and depending on the extent of the damage, to further events, including cell death. Evidence suggests that cell differentiation may also be a consequence of the DDR. During the formation of the infective hypha in the phytopathogenic fungus Ustilago maydis, two DDR kinases, Atr1 and Chk1, are required to induce a G2 cell cycle arrest, which in turn is essential to display the virulence program. However, the triggering factor of DDR in this process has remained elusive. In this report we provide data suggesting that no DNA damage is associated with the activation of the DDR during the formation of the infective filament in U. maydis. We have analyzed bulk DNA replication during the formation of the infective filament, and we found no signs of impaired DNA replication. Furthermore, using RPA-GFP fusion as a surrogate marker of the presence of DNA damage, we were unable to detect any sign of DNA damage at the cellular level. In addition, neither MRN nor 9-1-1 complexes, both instrumental to transmit the DNA damage signal, are required for the induction of the above mentioned cell cycle arrest, as well as for virulence. In contrast, we have found that the claspin-like protein Mrc1, which in other systems serves as scaffold for Atr1 and Chk1, was required for both processes. We discuss possible alternative ways to trigger the DDR, independent of DNA damage, in U. maydis during virulence program activation. © 2015 Tenorio-Gómez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


PubMed | Institute Biologia Funcional y Genomica CSIC
Type: Journal Article | Journal: PloS one | Year: 2015

DNA damage response (DDR) leads to DNA repair, and depending on the extent of the damage, to further events, including cell death. Evidence suggests that cell differentiation may also be a consequence of the DDR. During the formation of the infective hypha in the phytopathogenic fungus Ustilago maydis, two DDR kinases, Atr1 and Chk1, are required to induce a G2 cell cycle arrest, which in turn is essential to display the virulence program. However, the triggering factor of DDR in this process has remained elusive. In this report we provide data suggesting that no DNA damage is associated with the activation of the DDR during the formation of the infective filament in U. maydis. We have analyzed bulk DNA replication during the formation of the infective filament, and we found no signs of impaired DNA replication. Furthermore, using RPA-GFP fusion as a surrogate marker of the presence of DNA damage, we were unable to detect any sign of DNA damage at the cellular level. In addition, neither MRN nor 9-1-1 complexes, both instrumental to transmit the DNA damage signal, are required for the induction of the above mentioned cell cycle arrest, as well as for virulence. In contrast, we have found that the claspin-like protein Mrc1, which in other systems serves as scaffold for Atr1 and Chk1, was required for both processes. We discuss possible alternative ways to trigger the DDR, independent of DNA damage, in U. maydis during virulence program activation.

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