Job de Lange

The STAG1 and STAG2 paralogs are mutually exclusive subunits of cohesin, a large protein complex that controls different aspects of genome organization. Loss-of-function mutations in STAG2 are common in a variety of cancers, and have been associated with increased sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). This provides a possible entry point for therapeutic exploitation. However, insights in the underlying mechanism and additional determinants of this sensitivity are currently lacking. Here we show that STAG2-deficient cell lines exhibit variable PARPi sensitivity. While wildtype TP53 enhances PARPi sensitivity in RPE1 cells, STAG2 depletion does not trigger p53 or affect PARPi sensitivity in RPE1-TP53wt cells. PARPi sensitivity is determined by the capacity of STAG1 to compensate for the lack of STAG2. Thus, STAG2 is not uniquely required for the PARPi response, and its loss predominantly sensitizes cells through a general defect in cohesin function. PARP trapping underlies this sensitivity and is strongly influenced by basal PARP1 expression levels and further enhanced by oncogenic signaling. Perturbations in sister chromatid cohesion also enhances PARPi sensitivity, correlating with aggravated cohesion defects and increased chromosome breakage. Mechanistically, cohesin impairment does not appear to compromise repair of nickase-induced DNA lesions, suggesting that cohesin-defective cells are particularly vulnerable to a distinct class of lesions associated with trapped PARP. Together, our findings identify residual cohesin function and PARP1 expression as key determinants of PARPi response and suggest that successful clinical application of PARP inhibitors in STAG2-mutant cancers will require biomarker-guided patient stratification rather than STAG2 mutation status alone.

DDX11 is a DNA helicase involved in critical cellular functions, including DNA replication/repair/recombination as well as sister chromatid cohesion establishment. Bi-allelic mutations of DDX11 lead to Warsaw breakage syndrome (WABS), a rare genome instability disorder marked by significant prenatal and postnatal growth restriction, microcephaly, intellectual disability, and sensorineural hearing loss. The molecular mechanisms underlying WABS remain largely unclear. In this study, we uncover a novel role of DDX11 in regulating the macroautophagic/autophagic pathway. Specifically, we demonstrate that knockout of DDX11 in RPE-1 cells hinders the progression of autophagy. DDX11 depletion significantly reduces the conversion of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), suggesting a defect in autophagosome biogenesis. This is supported by imaging analysis with a LC3 reporter fused in tandem with the red and green fluorescent proteins (mRFP-GFP-LC3), which reveals fewer autophagosomes and autolysosomes in DDX11-knockout cells. Moreover, the defect in autophagosome biogenesis, observed in DDX11-depleted cells, is linked to an upstream impairment of the ATG16L1-precursor trafficking and maturation, a step critical to achieve the LC3 lipidation. Consistent with this, DDX11-lacking cells exhibit a diminished capacity to clear aggregates of a mutant HTT (huntingtin) N-terminal fragment fused to the green fluorescent protein (HTTQ74-GFP), an autophagy substrate. Finally, we demonstrate the occurrence of a functional interplay between DDX11 and SQSTM1, an autophagy cargo receptor protein, in supporting LC3 modification during autophagosome biogenesis. Our findings highlight a novel unprecedented function of DDX11 in the autophagy process with important implications for our understanding of WABS etiology.

The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.

The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by active DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, by using synthetic lethality CRISPR screens in isogenic human cell lines defective of specific cohesion regulators, we mapped the genetic dependencies induced by absence of DDX11 or ESCO2. The obtained high confidence synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identified the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravated cohesion defects in ESCO2 defective cells, leading to mitotic cell death. PAXIP1 promoted the global chromatin association of cohesin, independent of DNA replication, a function that could not be explained by indirect effects of PAXIP1 on transcription or the DNA damage response. Cohesin regulation by PAXIP1 required its binding partner PAGR1 and a conserved FDF motif in PAGR1. Similar motifs were previously found in multiple cohesin regulators, including CTCF, to mediate physical interactions with cohesin. PAXIP1 co-localizes with cohesin on multiple genomic loci, including at active gene promoters and enhancers. Together, this study identifies the PAXIP1-PAGR1 complex as a novel regulator of cohesin occupancy on chromatin. Possibly, this role in cohesin regulation is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.

The leading strand–oriented alternative PCNA clamp loader DSCC1-RFC functions in DNA replication, repair, and sister chromatid cohesion (SCC), but how it facilitates these processes is incompletely understood. Here, we confirm that loss of human DSCC1 results in reduced fork speed, increased DNA damage, and defective SCC. Genome-wide CRISPR screens in DSCC1-KO cells reveal multiple synthetically lethal interactions, enriched for DNA replication and cell cycle regulation. We show that DSCC1-KO cells require POLE3 for survival. Co-depletion of DSCC1 and POLE3, which both interact with the catalytic polymerase ε subunit, additively impair DNA replication, suggesting that these factors contribute to leading-strand DNA replication in parallel ways. An additional hit is MMS22L, which in humans forms a heterodimer with TONSL. Synthetic lethality of DSCC1 and MMS22L-TONSL likely results from detrimental SCC loss. We show that MMS22L-TONSL, like DDX11, functions in a SCC establishment pathway parallel to DSCC1-RFC. Because both DSCC1-RFC and MMS22L facilitate ESCO2 recruitment to replication forks, we suggest that distinct ESCO2 recruitment pathways promote SCC establishment following either cohesin conversion or de novo cohesin loading.

Budding uninhibited by benzimidazoles (BUB1) contributes to multiple mitotic processes. Here, we describe the first two patients with biallelic BUB1 germline mutations, who both display microcephaly, intellectual disability, and several patient-specific features. The identified mutations cause variable degrees of reduced total protein level and kinase activity, leading to distinct mitotic defects. Both patients’ cells show prolonged mitosis duration, chromosome segregation errors, and an overall functional spindle assembly checkpoint. However, while BUB1 levels mostly affect BUBR1 kinetochore recruitment, impaired kinase activity prohibits centromeric recruitment of Aurora B, SGO1, and TOP2A, correlating with anaphase bridges, aneuploidy, and defective sister chromatid cohesion. We do not observe accelerated cohesion fatigue. We hypothesize that unresolved DNA catenanes increase cohesion strength, with concomitant increase in anaphase bridges. In conclusion, BUB1 mutations cause a neurodevelopmental disorder, with clinical and cellular phenotypes that partially resemble previously described syndromes, including autosomal recessive primary microcephaly, mosaic variegated aneuploidy, and cohesinopathies.

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.

Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4^CSA ubiquitin ligase. How CRL4^CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4^CSA for optimal RNAPII ubiquitylation. Drug–genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.

Warsaw breakage syndrome (WABS), is caused by biallelic mutations of DDX11, a gene coding a DNA helicase. We have recently reported two affected sisters, compound heterozygous for a missense (p.Leu836Pro) and a frameshift (p.Lys303Glufs*22) variant. By investigating the pathogenic mechanism, we demonstrate the inability of the DDX11 p.Leu836Pro mutant to unwind forked DNA substrates, while retaining DNA binding activity. We observed the accumulation of patient‐derived cells at the G2/M phase and increased chromosomal fragmentation after mitomycin C treatment. The phenotype partially overlaps with features of the Fanconi anemia cells, which shows not only genomic instability but also defective mitochondria. This prompted us to examine mitochondrial functionality in WABS cells and revealed an altered aerobic metabolism. This opens the door to the further elucidation of the molecular and cellular basis of an impaired mitochondrial phenotype and sheds light on this fundamental process in cell physiology and the pathogenesis of these diseases.

Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DDX11 is essential for sister chromatid cohesion and resistance to G4 stabilizers. We propose that DDX11 is a DNA helicase protecting against G4 induced double-stranded breaks and concomitant loss of cohesion, possibly at DNA replication forks.