Exo1 phosphorylation inhibits exonuclease activity and prevents fork collapse in rad53 mutants independently of the 14-3-3 proteins

The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1–2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutantsSpanish Ministry of Economy and Competitiveness (MINECO) [FEDER-BFU2013-45182-P to C.M.C., M.S.]; University of Salamanca [KA6H/463AC01 to M.S.]; MINECO, AEI, Xunta de Galicia and FEDER [RYC-2012-10835, BFU2016-78121-P, ED431F-2016/019, ED431B-2016/016 to M.G.B.]; Junta de Castilla y León (JCyL), Program ‘Escalera de Excelencia’ [FEDER-CLU-2017-03]; JCyL Pre-doctoral Fellowship (to A.B.); MINECO Pre-Doctoral Fellowship (to E.C.M.); Xunta de Galicia Pre-doctoral Fellowship (ED481A-2018/041 to R.C.)S

Checkpoint inhibition of origin firing prevents DNA topological stress.

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors

Regulación de la replicación por el checkpoint de fase S en Saccharomyces cerevisiae

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.Para mantener la integridad genómica, el DNA debe ser protegido del daño provocado por agentes genotóxicos o por alteraciones surgidas durante la replicación. El checkpoint de fase S, mediado por Mec1 y Rad53 en Saccharomyces cerevisiae (ATR y Chk2 en células humanas, respectivamente) responde a perturbaciones en la replicación y coordina una respuesta celular global necesaria para mantener la integridad del genoma. Estamos interesados en entender cómo el checkpoint de fase S regula la replicación en estas condiciones, y más específicamente, en los mecanismos moleculares que preservan la estabilidad de las horquillas de replicación y promueven el reinicio de la síntesis de DNA tras bloqueos en la replicación. Como la quinasa Rad53 estabiliza las horquillas de replicación mediante un mecanismo dependiente de Exo1, hemos estudiado la regulación de esta nucleasa, y nuestros resultados indican que Exo1 es fosforilada por Rad53 en respuesta a estrés replicativo o daño en el DNA. Mutantes fosfomiméticos de EXO1 suprimen el colapso de horquillas de replicación de mutantes rad53, al igual que la deleción de la nucleasa, lo que sugiere fuertemente que la fosforilación de Exo1 tiene un efecto inhibitorio en la actividad de la proteína. Sin embargo, la deleción de EXO1 no suprime el defecto de reiniciación de horquillas observado en el mutante rad53, lo que sugiere que la reactivación de las horquillas bloqueadas es regulada mediante otro mecanismo. Nuestros resultados indican que Rad53 promueve el reinicio de horquillas de replicación a través de la inducción transcripcional de genes de respuesta a daño en el DNA. Por tanto, además de prevenir la degradación de las horquillas, Rad53 promueve el reinicio de la replicación induciendo la expresión de los genes de la ribonucleótido reductasa (RNR) y regulando la localización subcelular del complejo tras estrés replicativo.Peer Reviewe

Different nucleosomal landscapes at early and late replicating origins in Saccharomyces cerevisiae

Trabajo presentado al International Symposium "Cell Proliferation and Genome Integrity" organizado por la Fundación Ramón Areces y celebrado en Santander del 3 al 4 de abril de 2014.Eukaryotic genomes are replicated during S phase according to a temporal program. Several determinants seem to control the timing of origin firing, including the chromatin environment and epigenetic modifications. By performing high-resolution analysis of genome-wide nucleosome positioning we have identified different chromatin architectures at early and late replication origins. The different nucleosome patterns from early and late origins are already established in G1 and are tightly correlated with the genomic context. Moreover, specific early and late nucleosomal patterns seem to be fixed robustly, even in rpd3 mutants in which histone acetylation and origin timing have been significantly altered. Nevertheless, higher histone acetylation levels correlate with the local modulation of chromatinassociated features, leading to increased origin accessibility. In addition, we conducted parallel analyses of replication and nucleosome dynamics that revealed that origin conformation is modulated during origin activation.Peer reviewe

Checkpoint-dependent RNR induction promotes fork restart after replicative stress

The checkpoint kinase Rad53 is crucial to regulate DNA replication in the presence of replicative stress. Under conditions that interfere with the progression of replication forks, Rad53 prevents Exo1-dependent fork degradation. However, although EXO1 deletion avoids fork degradation in rad53 mutants, it does not suppress their sensitivity to the ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU). In this case, the inability to restart stalled forks is likely to account for the lethality of rad53 mutant cells after replication blocks. Here we show that Rad53 regulates replication restart through the checkpoint-dependent transcriptional response, and more specifically, through RNR induction. Thus, in addition to preventing fork degradation, Rad53 prevents cell death in the presence of HU by regulating RNR-expression and localization. When RNR is induced in the absence of Exo1 and RNR negative regulators, cell viability of rad53 mutants treated with HU is increased and the ability of replication forks to restart after replicative stress is restored.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2013-43766-P and Consolider Ingenio CSD2007-00015 to J.A.T.; BFU2010-20034 and BFU2013-45182-P to M.S.] and Cancer Researck UK [to J.D.]. E.C. is the recipient of a pre-doctoral fellowship from MINECO. M.S. was supported by a JAE-DOC contract from CSIC and by a Ramón y Cajal contract from MINECO (RYC-2009-04407).Peer Reviewe

Strand asymmetry of DNA damage tolerance mechanisms

19 p.-4 fig.DNA damage tolerance mechanisms are crucial for timely and accurate chromosomal replication in response to DNA polymerase stalling. Ubiquitylation of the replicative sliding clamp PCNA drives major tolerance pathways, error-free homologous recombination template switching and error-prone translesion synthesis, though their dynamics at forks and pathway choice determinants are poorly understood. Using strand-specific genomics we revealed an asymmetric nature of tolerance pathways, characterized by preferential template switching-driven recombinase engagement of stalled nascent lagging strands and translesion synthesis usage in response to leading strand polymerase stalling. This asymmetry, determined by a strand-dynamic interplay between PCNA-ubiquitin writers and erasers, likely stems from necessities dictated by leading and lagging strand replication mechanisms and has implications for asymmetric mutation inheritance.Spanish Ministry of Science grant PID2020-116003GB-100 (RB) Spanish Ministry of Science grant PID2019-109616GB-100 (AB, MPS) Junta de Castilla y León SA042P17 / SA103P20 (AB, RB) Junta de Castilla y León Postdoctoral Fellowship (JCC) Spanish Ministry of Science Formación del Personal Investigador PRE2018-084025 (DJS) Spanish Ministry of Science Juan de la Cierva - Incorporación Postdoctoral Fellowship IJC2019-041728-I (ECM) Junta de Castilla y León Predoctoral Fellowship (JZ) Swedish Research Council grant 2022-03478 (KS) MEXT grant 20H05940 (KS) AMED BINDS grant 22ama121020j0001 (KS)N

Transcriptomic profiles of cd47 in breast tumors predict outcome and are associated with immune activation

© 2021 by the authors.Targeting the innate immune system has attracted attention with the development of anti- CD47 antibodies. Anti-CD47 antibodies block the inhibition of the phagocytic activity of macrophages caused by the up-regulation of CD47 on tumor cells. In this study, public genomic data was used to identify genes highly expressed in breast tumors with elevated CD47 expression and analyzed the association between the presence of tumor immune infiltrates and the expression of the selected genes. We found that 142 genes positively correlated with CD47, of which 83 predicted favorable and 32 detrimental relapse-free survival (RFS). From those associated with favorable RFS, we selected the genes with immunologic biological functions and defined a CD47-immune signature composed of PTPRC, HLA-E, TGFBR2, PTGER4, ETS1, and OPTN. In the basal-like and HER2+ breast cancer subtypes, the expression of the CD47-immune signature predicted favorable outcome, correlated with the presence of tumor immune infiltrates, and with gene expression signatures of T cell activation. Moreover, CD47 up-regulated genes associated with favorable survival correlated with pro-tumoral macrophages. In summary, we described a CD47-immune gene signature composed of 6 genes associated with favorable prognosis, T cell activation, and pro-tumoral macrophages in breast cancer tumors expressing high levels of CD47.This work was supported by Instituto de Salud Carlos III (PI19/00808), ACEPAIN, Diputación de Albacete, CIBERONC and CRIS Cancer Foundation (to A.O.). Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the scientific foundation of the AECC and the CRIS Foundation (to A.P.). M.d.M.N.-L. was supported by the Spanish Ministry of Education (FPU grant; Ref.: FPU18/01319). The work carried out in our laboratories received support from the European Community through the regional development funding program (FEDER). B.G. was supported by the grants NVKP_16-1-2016-0037, 2018-1.3.1-VKE-2018-00032, and KH-129581

Transcriptomic Profiles of CD47 in Breast Tumors Predict Outcome and Are Associated with Immune Activation

Targeting the innate immune system has attracted attention with the development of anti- CD47 antibodies. Anti-CD47 antibodies block the inhibition of the phagocytic activity of macrophages caused by the up-regulation of CD47 on tumor cells. In this study, public genomic data was used to identify genes highly expressed in breast tumors with elevated CD47 expression and analyzed the association between the presence of tumor immune infiltrates and the expression of the selected genes. We found that 142 genes positively correlated with CD47, of which 83 predicted favorable and 32 detrimental relapse-free survival (RFS). From those associated with favorable RFS, we selected the genes with immunologic biological functions and defined a CD47-immune signature composed of PTPRC, HLA-E, TGFBR2, PTGER4, ETS1, and OPTN. In the basal-like and HER2+ breast cancer subtypes, the expression of the CD47-immune signature predicted favorable outcome, correlated with the presence of tumor immune infiltrates, and with gene expression signatures of T cell activation. Moreover, CD47 up-regulated genes associated with favorable survival correlated with pro-tumoral macrophages. In summary, we described a CD47-immune gene signature composed of 6 genes associated with favorable prognosis, T cell activation, and pro-tumoral macrophages in breast cancer tumors expressing high levels of CD47