The Interplay of Cohesin and the Replisome at Processive and Stressed DNA Replication Forks

The cohesin complex facilitates faithful chromosome segregation by pairing the sister chromatids after DNA replication until mitosis. In addition, cohesin contributes to proficient and error-free DNA replication. Replisome progression and establishment of sister chromatid cohesion are intimately intertwined processes. Here, we review how the key factors in DNA replication and cohesion establishment cooperate in unperturbed conditions and during DNA replication stress. We discuss the detailed molecular mechanisms of cohesin recruitment and the entrapment of replicated sister chromatids at the replisome, the subsequent stabilization of sister chromatid cohesion via SMC3 acetylation, as well as the role and regulation of cohesin in the response to DNA replication stress.

Bacillus subtilis RecA, DisA, and RadA/Sms Interplay Prevents Replication Stress by Regulating Fork Remodeling

Reviving Bacillus subtilis spores require the recombinase RecA, the DNA damage checkpoint sensor DisA, and the DNA helicase RadA/Sms to prevent a DNA replication stress. When a replication fork stalls at a template lesion, RecA filaments onto the lesion-containing gap and the fork is remodeled (fork reversal). RecA bound to single-strand DNA (ssDNA) interacts with and recruits DisA and RadA/Sms on the branched DNA intermediates (stalled or reversed forks), but DisA and RadA/Sms limit RecA activities and DisA suppresses its c-di-AMP synthesis. We show that RecA, acting as an accessory protein, activates RadA/Sms to unwind the nascent lagging-strand of the branched intermediates rather than to branch migrate them. DisA limits the ssDNA-dependent ATPase activity of RadA/Sms C13A, and inhibits the helicase activity of RadA/Sms by a protein-protein interaction. Finally, RadA/Sms inhibits DisA-mediated c-di-AMP synthesis and indirectly inhibits cell proliferation, but RecA counters this negative effect. We propose that the interactions among DisA, RecA and RadA/Sms, which are mutually exclusive, contribute to generate the substrate for replication restart, regulate the c-di-AMP pool and limit fork restoration in order to maintain cell survival.

Ploidy and recombination proficiency shape the evolutionary adaptation to constitutive DNA replication stress

In haploid budding yeast, evolutionary adaptation to constitutive DNA replication stress alters three genome maintenance modules: DNA replication, the DNA damage checkpoint, and sister chromatid cohesion. We asked how these trajectories depend on genomic features by comparing the adaptation in three strains: haploids, diploids, and recombination deficient haploids. In all three, adaptation happens within 1000 generations at rates that are correlated with the initial fitness defect of the ancestors. Mutations in individual genes are selected at different frequencies in populations with different genomic features, but the benefits these mutations confer are similar in the three strains, and combinations of these mutations reproduce the fitness gains of evolved populations. Despite the differences in the selected mutations, adaptation targets the same three functional modules despite differences in genomic features, revealing a common evolutionary response to constitutive DNA replication stress.

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular switch that enables the genome-protective function of RIF1 during DNA replication stress.

STEM-22. TARGETING REPLICATION STRESS RESPONSE FOR GLIOMA STEM CELL SPECIFIC CYTOTOXICITY

BACKGROUND Evidence suggests treatment resistant glioma stem cells (GSCs) drive glioblastoma (GBM) recurrence. Current treatments fail to eradicate GSC and novel GSC targeting therapies are a priority. GSC exhibit elevated DNA replication stress (RS) versus non GSC tumour cells driving constitutive DNA damage response (DDR) activation and efficient DNA repair. We previously demonstrated that targeting RS response with combined ATR and PARP inhibition (CAiPi) (VE821 and Olaparib) provides potent GSC specific cytotoxicity. In this study we investigated the underlying DDR phenotype which determines this vulnerability. RESULTS Paired GSC enriched (‘GSC’) and GSC depleted, differentiated (‘bulk’) populations were cultured from GBM specimens in neurobasal media with growth factors or serum containing media respectively. GSC exhibited reduced survival following exposure to CAiPi versus bulk. CAiPi significantly increased 53BP1 G1 phase nuclear bodies (53BP1NBs) in GSC, which are known to shield under-replicated DNA in actively transcribed genes. Mapping the genomic distribution of endogenously occurring replication dependent DNA double strand breaks via Breaks Ligation In Situ Sequencing (BLISS), revealed reduced intragenic DSB in long actively transcribed genes in GSC versus bulk at baseline, suggesting a reliance upon transcription coupled DSB repair in GSC. RNA seq demonstrated CAiPi-induced transcriptomic alterations in GSC including replication regulation and initiation. DNA fibre assay showed that CAiPi increased GSC new origin firing which correlated with PARP trapping. GSCs were rescued from CAiPi by roscovitine induced inhibition of excess origin firing. CAiPi is potently radiosensitizing by clonogenic assay and we demonstrated murine blood brain barrier penetration of CAiPi utilising VE822 and pamiparib in vivo. CONCLUSION Dysregulation of origin firing by CAiPi exposes a GSC specific vulnerability which results in DNA under-replication and abrogation of proficient DNA repair seen at long actively transcribed genes and has potential to be clinically translated as a GSC specific cytotoxic therapy.

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and Protein Phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2ACdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We showed that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1 that encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK ( cdc28F19) also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.

Targeting CDC7 potentiates ATR-CHK1 signaling inhibition through induction of DNA replication stress in liver cancer

Background Liver cancer is one of the most commonly diagnosed cancers and the fourth leading cause of cancer-related death worldwide. Broad-spectrum kinase inhibitors like sorafenib and lenvatinib provide only modest survival benefit to patients with hepatocellular carcinoma (HCC). This study aims to identify novel therapeutic strategies for HCC patients. Methods Integrated bioinformatics analyses and a non-biased CRISPR loss of function genetic screen were performed to identify potential therapeutic targets for HCC cells. Whole-transcriptome sequencing (RNA-Seq) and time-lapse live imaging were performed to explore the mechanisms of the synergy between CDC7 inhibition and ATR or CHK1 inhibitors in HCC cells. Multiple in vitro and in vivo assays were used to validate the synergistic effects. Results Through integrated bioinformatics analyses using the Cancer Dependency Map and the TCGA database, we identified ATR-CHK1 signaling as a therapeutic target for liver cancer. Pharmacological inhibition of ATR or CHK1 leads to robust proliferation inhibition in liver cancer cells having a high basal level of replication stress. For liver cancer cells that are resistant to ATR or CHK1 inhibition, treatment with CDC7 inhibitors induces strong DNA replication stress and consequently such drugs show striking synergy with ATR or CHK1 inhibitors. The synergy between ATR-CHK1 inhibition and CDC7 inhibition probably derives from abnormalities in mitosis inducing mitotic catastrophe. Conclusions Our data highlights the potential of targeting ATR-CHK1 signaling, either alone or in combination with CDC7 inhibition, for the treatment of liver cancer.