Search

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.

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.

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 mutants

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 mutants.

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.

[Background]: Eukaryotic genomes are replicated during S phase according to a temporal program. Several determinants control the timing of origin firing, including the chromatin environment and epigenetic modifications. However, how chromatin structure influences the timing of the activation of specific origins is still poorly understood. [Results]: By performing high-resolution analysis of genome-wide nucleosome positioning we have identified different chromatin architectures at early and late replication origins. These different patterns are already established in G1 and are tightly correlated with the organization of adjacent transcription units. Moreover, specific early and late nucleosomal patterns are 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 chromatin structure, leading to increased origin accessibility. In addition, we conducted parallel analyses of replication and nucleosome dynamics that revealed that chromatin structure at origins is modulated during origin activation. [Conclusions]: Our results show that early and late replication origins present distinctive nucleosomal configurations, which are preferentially associated to different genomic regions. Our data also reveal that origin structure is dynamic and can be locally modulated by histone deacetylation, as well as by origin activation. These data offer novel insight into the contribution of chromatin structure to origin selection and firing in budding yeast.

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.