Highlights d Cohesion loss is a common feature of cancer cells d DNA replication stress induces cohesion loss d The cohesin remover WAPL is essential in replication stress conditions d WAPL promotes repair and restart of a broken replication fork

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.

ranscription of protein-coding and noncoding genes requires RNA polymerase II (RNAPII), which synthesizes RNA transcripts complementary to the DNA template strand. The presence of DNA lesions in the template strand causes stalling of elongating RNAPII (RNAPIIo), which leads to genome-wide transcriptional arrest 1-3 . It is essential that cells overcome this arrest and restore transcription. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions through the proteins CSB, CSA and UVSSA [4][5][6] . Inactivating mutations in CSB and CSA cause Cockayne syndrome (CS), which is characterized by severe neurological dysfunction related to persistent RNAPII arrest at DNA lesions 2,7 .The sequential and cooperative actions of CSB, CSA and UVSSA recruit TFIIH to DNA damage-stalled RNAPII to initiate DNA repair 6 . In addition to protein-protein contacts, efficient transfer of TFIIH onto RNAPII requires ubiquitylation of a single lysine on the largest subunit of RNAPII (RPB1-K1268), which is essential for efficient TCR 2 . This DNA damage-induced modification of RNAPII is dependent on cullin-ring type E3-ligases (CRLs) and is strongly decreased in CSA-deficient cells 2 , which indicates that the CRL4 CSA E3 ligase complex drives RNAPII ubiquitylation.CSB binds to DNA upstream of RNAPII 8 (Extended Data Fig. 1a) and recruits the CRL4 CSA complex through an evolutionarily conserved motif in its carboxy terminus 6 . However, how the activity of CRL4 CSA ubiquitin ligase is specifically directed towards the K1268 site remains to be elucidated. Results A CRISPR screen identifies ELOF1 as a putative TCR gene.To identify unknown TCR genes, we performed a genome-wide CRISPR screen in the presence of the compound illudin S, which induces transcription-blocking DNA lesions that are eliminated by TCR 9 . RPE1-iCas9 cells were transduced with the pLCKO-TKOv3 library, which contains 70,948 single guide RNAs (sgRNAs) targeting open reading frames 10 , and cultured for 12 population doublings, after which sgRNA contents were analysed (Extended Data Fig. 1b).Using a false-discovery rate (FDR) cut-off of 0.01, we found 104 sensitizer hits and 18 hits conferring resistance to illudin S. The strongest resistance was conferred by guide RNAs (gRNAs) targeting PTGR1, which is in line with its known role in bioactivating illudin S 11 (Fig. 1a and Extended Data Fig. 1c). Nine known core TCR genes, including CSB, CSA and UVSSA, but also genes connected to transcription recovery after UV irradiation (HIRA 12 , DOT1L 13 and STK19 (ref. 14 ) (Fig. 1a,b), were required for illudin S tolerance. Consistent with known effects of illudin S on replication 9 , we found the 9-1-1 complex, translesion synthesis and sister-chromatid cohesion components (Fig. 1b). Our screen also identified the ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation Yana van der Weegen 1,10 , Klaas de Lint 2,10 , Diana van den Heuvel

In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABSderived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated.

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.

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.

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.

In order to duplicate, cells need to faithfully replicate their DNA followed by the coordinated transfer of duplicated chromosomes to the future daughter cells. To ensure correct segregation, sister chromatids are paired by the cohesin complex from the moment of their synthesis until their separation in mitosis. Defects in cohesin function and mutations in cohesin genes are associated with cancer and are the cause of developmental disorders called cohesinopathies. In this thesis, we aimed to enhance the understanding of the molecular mechanisms underlying sister chromatid cohesion biology to gain insights relevant for cohesinopathies and cancer. By studying Warsaw Breakage Syndrome patient derived mutant DDX11, we reveal that sister chromatid cohesion establishment and DNA replication stress response depend on the helicase function of DDX11. Interestingly, the all studied patients have decreased, but residual, functional DDX11. Together with the finding that DDX11 loss leads to P53 dependent proliferation defects and mice with helicase defective DDX11 are inviable, this suggests complete loss of functional DDX11 is not compatible with life. In addition, we show that cancer cells of various origins often exhibit cohesion defects. In untransformed cells induction of DNA replication stress is sufficient to trigger sister chromatid cohesion loss. Furthermore, we unveil that the cohesin antagonist WAPL is necessary to deal with replication stress by promoting the repair of broken replication forks. Our data suggests WAPL-dependent cohesin removal at stalled replication forks may contribute to sister chromatid cohesion defects in cancer cells. Both the requirement for WAPL and the cohesion loss phenotype may provide specific vulnerabilities of cancer cells that may be targeted. To expose vulnerabilities of cells with cohesion defects and identify novel genes involved in sister chromatid cohesion, we performed genome-wide CRISPR screens in cohesion defective cells. This revealed multiple synthetic lethal interactions, enriched in genes involved in DNA replication and mitosis. Among these, we identify a role for MMS22L-TONSL in sister chromatid cohesion establishment. Furthermore, we identify a novel regulator of cohesin occupancy on chromatin, the PAXIP1-PAGR1 heterodimer. PAXIP1 co-localizes with cohesin on multiple genomic loci, including at active promoters and enhancers, thereby likely contributing to multiple cohesin related functions. Together, our work increases the understanding of the molecular pathways contributing to sister chromatid cohesion establishment. These findings may have implications for the development of therapeutic strategies for cancer and provide insight in the etiology of cohesinopathies.