SMARCAL1 is a dual regulator of innate immune signaling and PD-L1 expression that promotes tumor immune evasion.

Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy.

Replication stress impairs chromosome segregation and preimplantation development in human embryos.

Human cleavage-stage embryos frequently acquire chromosomal aneuploidies during mitosis due to unknown mechanisms. Here, we show that S phase at the 1-cell stage shows replication fork stalling, low fork speed, and DNA synthesis extending into G2 phase. DNA damage foci consistent with collapsed replication forks, DSBs, and incomplete replication form in G2 in an ATR- and MRE11-dependent manner, followed by spontaneous chromosome breakage and segmental aneuploidies. Entry into mitosis with incomplete replication results in chromosome breakage, whole and segmental chromosome errors, micronucleation, chromosome fragmentation, and poor embryo quality. Sites of spontaneous chromosome breakage are concordant with sites of DNA synthesis in G2 phase, locating to gene-poor regions with long neural genes, which are transcriptionally silent at this stage of development. Thus, DNA replication stress in mammalian preimplantation embryos predisposes gene-poor regions to fragility, and in particular in the human embryo, to the formation of aneuploidies, impairing developmental potential.

Functional interrogation of DNA damage response variants with base editing screens.

Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.

Oncogenic IDH mutations increase heterochromatin-related replication stress without impacting homologous recombination.

Oncogenic mutations in isocitrate dehydrogenases 1 and 2 (IDH1/2) produce 2-hydroxyglutarate (2HG), which inhibits dioxygenases that modulate chromatin dynamics. The effects of 2HG have been reported to sensitize IDH tumors to poly-(ADP-ribose) polymerase (PARP) inhibitors. However, unlike PARP-inhibitor-sensitive BRCA1/2 tumors, which exhibit impaired homologous recombination, IDH-mutant tumors have a silent mutational profile and lack signatures associated with impaired homologous recombination. Instead, 2HG-producing IDH mutations lead to a heterochromatin-dependent slowing of DNA replication accompanied by increased replication stress and DNA double-strand breaks. This replicative stress manifests as replication fork slowing, but the breaks are repaired without a significant increase in mutation burden. Faithful resolution of replicative stress in IDH-mutant cells is dependent on poly-(ADP-ribosylation). Treatment with PARP inhibitors increases DNA replication but results in incomplete DNA repair. These findings demonstrate a role for PARP in the replication of heterochromatin and further validate PARP as a therapeutic target in IDH-mutant tumors.

CRISPR-based genome editing through the lens of DNA repair.

Genome editing technologies operate by inducing site-specific DNA perturbations that are resolved by cellular DNA repair pathways. Products of genome editors include DNA breaks generated by CRISPR-associated nucleases, base modifications induced by base editors, DNA flaps created by prime editors, and integration intermediates formed by site-specific recombinases and transposases associated with CRISPR systems. Here, we discuss the cellular processes that repair CRISPR-generated DNA lesions and describe strategies to obtain desirable genomic changes through modulation of DNA repair pathways. Advances in our understanding of the DNA repair circuitry, in conjunction with the rapid development of innovative genome editing technologies, promise to greatly enhance our ability to improve food production, combat environmental pollution, develop cell-based therapies, and cure genetic and infectious diseases.

Histone methylation antagonism drives tumor immune evasion in squamous cell carcinomas.

How cancer-associated chromatin abnormalities shape tumor-immune interaction remains incompletely understood. Recent studies have linked DNA hypomethylation and de-repression of retrotransposons to anti-tumor immunity through the induction of interferon response. Here, we report that inactivation of the histone H3K36 methyltransferase NSD1, which is frequently found in squamous cell carcinomas (SCCs) and induces DNA hypomethylation, unexpectedly results in diminished tumor immune infiltration. In syngeneic and genetically engineered mouse models of head and neck SCCs, NSD1-deficient tumors exhibit immune exclusion and reduced interferon response despite high retrotransposon expression. Mechanistically, NSD1 loss results in silencing of innate immunity genes, including the type III interferon receptor IFNLR1, through depletion of H3K36 di-methylation (H3K36me2) and gain of H3K27 tri-methylation (H3K27me3). Inhibition of EZH2 restores immune infiltration and impairs the growth of Nsd1-mutant tumors. Thus, our work uncovers a druggable chromatin cross talk that regulates the viral mimicry response and enables immune evasion of DNA hypomethylated tumors.

REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.

BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.

HATtracting Nucleases to Stalled Forks.

In this issue of Molecular Cell, Kim et al., 2020 report that PCAF is a fork-associated histone acetyltransferase (HAT) that regulates the stability of stalled forks and the response to PARP inhibition in BRCA1/2-deficient cells.

Extrachromosomal telomere DNA derived from excessive strand displacements.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

The BRCT Domains of the BRCA1 and BARD1 Tumor Suppressors Differentially Regulate Homology-Directed Repair and Stalled Fork Protection.

The BRCA1 tumor suppressor preserves genome integrity through both homology-directed repair (HDR) and stalled fork protection (SFP). In vivo, BRCA1 exists as a heterodimer with the BARD1 tumor suppressor, and both proteins harbor a phosphate-binding BRCT domain. Here, we compare mice with mutations that ablate BRCT phospho-recognition by Bard1 (Bard1S563F and Bard1K607A) or Brca1 (Brca1S1598F). Brca1S1598F abrogates both HDR and SFP, suggesting that both pathways are likely impaired in most BRCA1 mutant tumors. Although not affecting HDR, the Bard1 mutations ablate poly(ADP-ribose)-dependent recruitment of BRCA1/BARD1 to stalled replication forks, resulting in fork degradation and chromosome instability. Nonetheless, Bard1S563F/S563F and Bard1K607A/K607A mice, unlike Brca1S1598F/S1598F mice, are not tumor prone, indicating that HDR alone is sufficient to suppress tumor formation in the absence of SFP. Nevertheless, because SFP, unlike HDR, is also impaired in heterozygous Brca1/Bard1 mutant cells, SFP and HDR may contribute to distinct stages of tumorigenesis in BRCA1/BARD1 mutation carriers.

CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.

Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.

Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments.

Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.

Restoration of Replication Fork Stability in BRCA1- and BRCA2-Deficient Cells by Inactivation of SNF2-Family Fork Remodelers.

To ensure the completion of DNA replication and maintenance of genome integrity, DNA repair factors protect stalled replication forks upon replication stress. Previous studies have identified a critical role for the tumor suppressors BRCA1 and BRCA2 in preventing the degradation of nascent DNA by the MRE11 nuclease after replication stress. Here we show that depletion of SMARCAL1, a SNF2-family DNA translocase that remodels stalled forks, restores replication fork stability and reduces the formation of replication stress-induced DNA breaks and chromosomal aberrations in BRCA1/2-deficient cells. In addition to SMARCAL1, other SNF2-family fork remodelers, including ZRANB3 and HLTF, cause nascent DNA degradation and genomic instability in BRCA1/2-deficient cells upon replication stress. Our observations indicate that nascent DNA degradation in BRCA1/2-deficient cells occurs as a consequence of MRE11-dependent nucleolytic processing of reversed forks generated by fork remodelers. These studies provide mechanistic insights into the processes that cause genome instability in BRCA1/2-deficient cells.

Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity.

DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.

Stressing Out About RAD52.

The role of mammalian RAD52 has been mysterious due to the lack of a strong DNA repair phenotype of RAD52-deficient cells. In this issue of Molecular Cell, studies by Bhowmick et al. (2016) and Sotiriou et al. (2016) reveal an unexpected role for RAD52 in promoting DNA synthesis following replication stress.

Strand annealing and motor driven activities of SMARCAL1 and ZRANB3 are stimulated by RAD51 and the paralog complex.

SMARCAL1, ZRANB3 and HLTF are required for the remodeling of replication forks upon stress to promote genome stability. RAD51, along with the RAD51 paralog complex, were also found to have recombination-independent functions in fork reversal, yet the underlying mechanisms remained unclear. Using reconstituted reactions, we build upon previous data to show that SMARCAL1, ZRANB3 and HLTF have unequal biochemical capacities, explaining why they have non-redundant functions. SMARCAL1 uniquely anneals RPA-coated ssDNA, which depends on its direct interaction with RPA, but not on ATP. SMARCAL1, along with ZRANB3, but not HLTF efficiently employ ATPase driven translocase activity to rezip RPA-covered bubbled DNA, which was proposed to mimic elements of fork reversal. In contrast, ZRANB3 and HLTF but not SMARCAL1 are efficient in branch migration that occurs downstream in fork remodeling. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2), directly stimulate the motor-driven activities of SMARCAL1 and ZRANB3 but not HLTF, and the interplay is underpinned by physical interactions. Our data provide a possible mechanism explaining previous cellular experiments implicating RAD51 and BCDX2 in fork reversal.

Polyubiquitinated PCNA recruits the ZRANB3 translocase to maintain genomic integrity after replication stress.

Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.

The DNA damage response: making it safe to play with knives.

Damage to our genetic material is an ongoing threat to both our ability to faithfully transmit genetic information to our offspring as well as our own survival. To respond to these threats, eukaryotes have evolved the DNA damage response (DDR). The DDR is a complex signal transduction pathway that has the ability to sense DNA damage and transduce this information to the cell to influence cellular responses to DNA damage. Cells possess an arsenal of enzymatic tools capable of remodeling and repairing DNA; however, their activities must be tightly regulated in a temporal, spatial, and DNA lesion-appropriate fashion to optimize repair and prevent unnecessary and potentially deleterious alterations in the structure of DNA during normal cellular processes. This review will focus on how the DDR controls DNA repair and the phenotypic consequences of defects in these critical regulatory functions in mammals.

A genome-wide camptothecin sensitivity screen identifies a mammalian MMS22L-NFKBIL2 complex required for genomic stability.

Replication stress involving collision of replisomes with camptothecin (CPT)-stabilized DNA-Topoisomerase I adducts activates an ATR-dependent pathway to promote repair by homologous recombination. To identify human genes that protect cells from such replication stress, we performed a genome-wide CPT sensitivity screen. Among numerous candidate genes are two previously unstudied proteins: the ankyrin repeat protein NFKBIL2 and C6ORF167 (MMS22L), distantly related to yeast replication stress regulator Mms22p. MMS22L and NFKBIL2 interact with each other and with FACT (facilitator of chromatin transcription) and MCM (minichromosome maintenance) complexes. Cells depleted of NFKBIL2 or MMS22L are sensitive to DNA-damaging agents, load phosphorylated RPA onto chromatin in a CTIP-dependent manner, activate the ATR/ATRIP-CHK1 and double-strand break repair signaling pathways, and are defective in HR. This study identifies MMS22L-NFKBIL2 as components of the replication stress control pathway and provides a resource for discovery of additional components of this pathway.

Treacher Collins syndrome TCOF1 protein cooperates with NBS1 in the DNA damage response.

The signal transduction pathway of the DNA damage response (DDR) is activated to maintain genomic integrity following DNA damage. The DDR promotes genomic integrity by regulating a large network of cellular activities that range from DNA replication and repair to transcription, RNA splicing, and metabolism. In this study we define an interaction between the DDR factor NBS1 and TCOF1, a nucleolar protein that regulates ribosomal DNA (rDNA) transcription and is mutated in Treacher Collins syndrome. We show that NBS1 relocalizes to nucleoli after DNA damage in a manner dependent on TCOF1 and on casein kinase II and ATM, which are known to modify TCOF1 by phosphorylation. Moreover, we identify a putative ATM phosphorylation site that is required for NBS1 relocalization to nucleoli in response to DNA damage. Last, we report that TCOF1 promotes cellular resistance to DNA damaging agents. Collectively, our findings identify TCOF1 as a DDR factor that could cooperate with ATM and NBS1 to suppress inappropriate rDNA transcription and maintain genomic integrity after DNA damage.