Role of protein phosphatases PP1, PP2A, PP4 and Cdc14 in the DNA damage response

Maintenance of genome integrity is fundamental for cellular physiology. Our hereditary information encoded in the DNA is intrinsically susceptible to suffer variations, mostly due to the constant presence of endogenous and environmental genotoxic stresses. Genomic insults must be repaired to avoid loss or inappropriate transmission of the genetic information, a situation that could lead to the appearance of developmental anomalies and tumorigenesis. To safeguard our genome, cells have evolved a series of mechanisms collectively known as the DNA damage response (DDR). This surveillance system regulates multiple features of the cellular response, including the detection of the lesion, a transient cell cycle arrest and the restoration of the broken DNA molecule. While the role of multiple kinases in the DDR has been well documented over the last years, the intricate roles of protein dephosphorylation have only recently begun to be addressed. In this review, we have compiled recent information about the function of protein phosphatases PP1, PP2A, PP4 and Cdc14 in the DDR, focusing mainly on their capacity to regulate the DNA damage checkpoint and the repair mechanism encompassed in the restoration of a DNA lesion

Sgs1’s roles in DNA end resection, HJ dissolution, and crossover suppression require a two-step SUMO regulation dependent on Smc5/6

The RecQ helicase Sgs1 plays critical roles during DNA repair by homologous recombination, fromend resection to Holliday junction (HJ) dissolution. Sgs1 has both pro- and anti-recombinogenic roles, and therefore its activity must be tightly regulated. However, the controls involved in recruitment and activation of Sgs1 at damaged sites are unknown. Here we show a two-step role for Smc5/6 in recruiting and activating Sgs1 through SUMOylation. First, auto-SUMOylation of Smc5/6 subunits leads to recruitment of Sgs1 as part of the STR (Sgs1–Top3–Rmi1) complex, mediated by two SUMO-interacting motifs (SIMs) on Sgs1 that specifically recognize SUMOylated Smc5/6. Second, Smc5/6-dependent SUMOylation of Sgs1 and Top3 is required for the efficient function of STR. Sgs1 mutants impaired in recognition of SUMOylated Smc5/6 (sgs1-SIMΔ) or SUMO-dead alleles (sgs1-KR) exhibit unprocessed HJs at damaged replication forks, increased crossover frequencies during double-strand break repair, and severe impairment in DNA end resection. Smc5/6 is a key regulator of Sgs1’s recombination functions.We thank the Aragon laboratory for discussions and critical reading of the manuscript.We thank the Clinical Sciences Centre Proteomics Facility (P. Cutillas and P. Faull) for help and advice on our proteomic analysis. Work in J.T.-R.’s laboratory is supported by grants BFU2015-71308-P and BFU2013-50245-EXP from the Spanish Ministry of Economy and Competitivity.Work in the Aragon laboratory was supported by the intramural programme of the Medical Research Council UK and the Wellcome Trust (100955)

The NDR/LATS Kinase Cbk1 Controls the Activity of the Transcriptional Regulator Bcr1 during Biofilm Formation in Candida albicans

In nature, many microorganisms form specialized complex, multicellular, surface-attached communities called biofilms. These communities play critical roles in microbial pathogenesis. The fungal pathogen Candida albicans is associated with catheter-based infections due to its ability to establish biofilms. The transcription factor Bcr1 is a master regulator of C. albicans biofilm development, although the full extent of its regulation remains unknown. Here, we report that Bcr1 is a phosphoprotein that physically interacts with the NDR kinase Cbk1 and undergoes Cbk1-dependent phosphorylation. Mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to alanine markedly impaired Bcr1 function during biofilm formation and virulence in a mouse model of disseminated candidiasis. Cells lacking Cbk1, or any of its upstream activators, also had reduced biofilm development. Notably, mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to glutamate in cbk1Δ cells upregulated the transcription of Bcr1-dependent genes and partially rescued the biofilm defects of a cbk1Δ strain. Therefore, our data uncovered a novel role of the NDR/LATS kinase Cbk1 in the regulation of biofilm development through the control of Bcr1

Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription

[EN]Kinases and phosphatases regulate messenger RNA synthesis through post-translational modi cation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. In yeast, the phosphatase Cdc14 is required for mitotic exit and for segregation of repetitive regions4. Cdc14 is also a subunit of the silencing complex RENT, but no roles in transcriptional repression have been described. Here we report that inactivation of Cdc14 causes silencing defects at the intergenic spacer sequences of ribosomal genes during interphase and at Y0 repeats in subtelomeric regions during mitosis. We show that the role of Cdc14 in silencing is independent of the RENT deacetylase subunit Sir2. Instead, Cdc14 acts directly on RNA polymerase II by targeting CTD phosphorylation at Ser 2 and Ser 5. We also find that the role of Cdc14 as a CTD phosphatase is conserved in humans. Finally, telomere segregation defects in cdc14 mutants4 correlate with the presence of subtelomeric Y0 elements and can be rescued by transcriptional inhibition of RNA polymerase II

The double-edge sword of the DNA damage response

Trabajo presentado en la 11º Reunión de la Red Española de Levaduras, celebrada en El Escorial (España), del 13 al 15 de diciembre de 2017Peer reviewe

Linking spindle stability and DNA repair

Resumen de la conferencia presentada en el Barcelona BioMed Seminars el 17 de febrero de 2016.Endogenous or exogenous agents that cause genotoxic stress are constantly threatening the genomes of all organisms. In response to a DNA lesion, different processes (collectively known as DDR) are triggered in order to coordinate the repair of the damage with cell cycle progression. This signal is driven by phosphorylation events that activate both the repair of the broken DNA and the DNA damage checkpoint arrest. While phosphorylation events after DNA damage has been thoroughly studied in DDR, little is known about the role of dephosphorlation during the response. Recently, the Cdc14 phosphatase has been implicated as a key regulator of the DDR. In fact, previous results coming from our laboratory have demonstrated that this phosphatase is required to promote recombinatorial repair. However, its exact molecular function in DNA repair largely remains to be established. Here we show that in response to a DSB (double strand break) induced by the expression of the HO endonuclease, Cdc14 is release from the nucleolus and relocalize to the SPBs (Spindle Pole Bodies). We have identified Spc110, the intranuclear receptor for the ¿-tubulin complex, as a putative target of the phosphatase during the DNA damage response. After induction of a DSB, Cdc14 counterbalance the phosphorylation imposed by the Cdk over Spc110. This steady state of Spc110 phosphorylation is required for the appropriate alignment of the metaphase spindle along the bud axis of the mother cell, as well as to recruit the DSB generated to one of the SPBs. Surprisingly, disruption of the metaphase spindle impair DSB-SPB tethering and DSB repair by homologous recombination. Together, our results point to the function of Cdc14 in DNA repair by stabilizing the metaphase spindle during the formation of a DNA lesion and and suggest that the relocation of damage sites to the SPBs plays an important role in a naturally occurring repair process that minimizes genome instability.Peer Reviewe

Nucleolar condensation: a new mechanism to control mitotic exit

The nucleolus in Saccharomyces cerevisiae is one of the last genomic regions to be condensed in mitosis. A new study shows that this extended nucleolar relaxation state is fundamental for the timely execution of mitotic exit.Peer reviewe

Genome-wide analysis of DNA repair at single-nucleotide resolution

Poster de la conferencia celebrada el 22 de enero de 2020, dentro de la Conference series cabimer 2020-202

The Yin and Yang of the DNA damage response

Trabajo presentado en el RED-CONSOLIDER Meeting: Genomic Instability, celebrado en Lleida (España), del 18 al 20 de febrero de 201