Nascent DNA sequencing and its diverse applications in genome integrity research.

Multiple DNA repair pathways and biological responses to DNA damage have evolved to protect cells from various types of lesions to which they are subjected. Although DNA repair systems are mechanistically distinct, all process the damaged region and then insert new bases to fill the gap. In 1969, Robert Painter developed an assay called "unscheduled" DNA synthesis (UDS), which measures DNA repair synthesis as the uptake of radiolabeled DNA precursors distinct from replicative synthesis. Contemporary detection of nascent DNA during repair by next-generation sequencing grants genome-wide information about the nature of lesions that threaten genome integrity. Recently, we developed the SAR-seq (synthesis associated with repair sequencing) method, which provides a high-resolution view of UDS. SAR-seq has been utilized to map programmed DNA repair sites in non-dividing neurons, replication initiation zones, monitor 53BP1 function in countering end-resection, and to identify regions of the genome that fail to complete replication during S phase but utilize repair synthesis during mitosis (MiDAS). As an example of SAR-seq, we present data showing that sites replicated during mitosis correspond to common fragile sites, which have been linked to tumor progression, cellular senescence, and aging.

EXO1 protects BRCA1-deficient cells against toxic DNA lesions.

Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and cancer. Importantly, BRCA1/2 deficiency also causes therapeutically targetable vulnerabilities. Here, we identify the dependency on the end resection factor EXO1 as a key vulnerability of BRCA1-deficient cells. EXO1 deficiency generates poly(ADP-ribose)-decorated DNA lesions during S phase that associate with unresolved DSBs and genomic instability in BRCA1-deficient but not in wild-type or BRCA2-deficient cells. Our data indicate that BRCA1/EXO1 double-deficient cells accumulate DSBs due to impaired repair by single-strand annealing (SSA) on top of their HR defect. In contrast, BRCA2-deficient cells retain SSA activity in the absence of EXO1 and hence tolerate EXO1 loss. Consistent with a dependency on EXO1-mediated SSA, we find that BRCA1-mutated tumors show elevated EXO1 expression and increased SSA-associated genomic scars compared with BRCA1-proficient tumors. Overall, our findings uncover EXO1 as a promising therapeutic target for BRCA1-deficient tumors.

SOD1 is a synthetic lethal target in PPM1D-mutant leukemia cells.

The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase, Mg2+/Mn2+ dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacologic target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate the protective role of SOD1 against oxidative stress in PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.

Comprehensive mapping of cell fates in microsatellite unstable cancer cells supports dual targeting of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.

Comprehensive mapping of cell fates in microsatellite unstable cancer cells support dual targe6ng of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN, knowledge that would be helpful for informing clinical development of WRN-targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system wherein the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We find that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we find no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provided the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggested a potential therapeutical rationale for dual targeting of WRN and ATR.

Active DNA demethylation promotes cell fate specification and the DNA damage response.

Neurons harbor high levels of single-strand DNA breaks (SSBs) that are targeted to neuronal enhancers, but the source of this endogenous damage remains unclear. Using two systems of postmitotic lineage specification-induced pluripotent stem cell-derived neurons and transdifferentiated macrophages-we show that thymidine DNA glycosylase (TDG)-driven excision of methylcytosines oxidized with ten-eleven translocation enzymes (TET) is a source of SSBs. Although macrophage differentiation favors short-patch base excision repair to fill in single-nucleotide gaps, neurons also frequently use the long-patch subpathway. Disrupting this gap-filling process using anti-neoplastic cytosine analogs triggers a DNA damage response and neuronal cell death, which is dependent on TDG. Thus, TET-mediated active DNA demethylation promotes endogenous DNA damage, a process that normally safeguards cell identity but can also provoke neurotoxicity after anticancer treatments.

A genetic roadmap to the response to genotoxic agents in human cells.

To maintain genome fidelity and prevent diseases such as cancer, our cells must constantly detect, and efficiently and precisely repair, DNA damage. Paradoxically, DNA-damaging agents in the form of radiation and chemotherapy are also used to treat cancer. Olivieri et al. used a CRISPR-based screen to identify genes that, when disrupted, lead to sensitivity or resistance to 27 different DNA-damaging agents used in the lab and/or in the clinic to treat cancer patients1. Their results reveal multiple new genes and connections that regulate these critical DNA damage repair pathways, with implications for basic and clinical research as well as cancer therapy.

S1-END-seq reveals DNA secondary structures in human cells.

DNA becomes single stranded (ssDNA) during replication, transcription, and repair. Transiently formed ssDNA segments can adopt alternative conformations, including cruciforms, triplexes, and quadruplexes. To determine whether there are stable regions of ssDNA in the human genome, we utilized S1-END-seq to convert ssDNA regions to DNA double-strand breaks, which were then processed for high-throughput sequencing. This approach revealed two predominant non-B DNA structures: cruciform DNA formed by expanded (TA)n repeats that accumulate in microsatellite unstable human cancer cell lines and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine mirror repeats common across a variety of cell lines. We show that H-DNA is enriched during replication, that its genomic location is highly conserved, and that H-DNA formed by (GAA)n repeats can be disrupted by treatment with a (GAA)n-binding polyamide. Finally, we show that triplex-forming repeats are hotspots for mutagenesis. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability.

Disrupted control of origin activation compromises genome integrity upon destabilization of Polε and dysfunction of the TRP53-CDKN1A/P21 axis.

The maintenance of genome stability relies on coordinated control of origin activation and replication fork progression. How the interplay between these processes influences human genetic disease and cancer remains incompletely characterized. Here we show that mouse cells featuring Polε instability exhibit impaired genome-wide activation of DNA replication origins, in an origin-location-independent manner. Strikingly, Trp53 ablation in primary Polε hypomorphic cells increased Polε levels and origin activation and reduced DNA damage in a transcription-dependent manner. Transcriptome analysis of primary Trp53 knockout cells revealed that the TRP53-CDKN1A/P21 axis maintains appropriate levels of replication factors and CDK activity during unchallenged S phase. Loss of this control mechanism deregulates origin activation and perturbs genome-wide replication fork progression. Thus, while our data support an impaired origin activation model for genetic diseases affecting CMG formation, we propose that loss of the TRP53-CDKN1A/P21 tumor suppressor axis induces inappropriate origin activation and deregulates genome-wide fork progression.

Human topoisomerases and their roles in genome stability and organization.

Human topoisomerases comprise a family of six enzymes: two type IB (TOP1 and mitochondrial TOP1 (TOP1MT), two type IIA (TOP2A and TOP2B) and two type IA (TOP3A and TOP3B) topoisomerases. In this Review, we discuss their biochemistry and their roles in transcription, DNA replication and chromatin remodelling, and highlight the recent progress made in understanding TOP3A and TOP3B. Because of recent advances in elucidating the high-order organization of the genome through chromatin loops and topologically associating domains (TADs), we integrate the functions of topoisomerases with genome organization. We also discuss the physiological and pathological formation of irreversible topoisomerase cleavage complexes (TOPccs) as they generate topoisomerase DNA-protein crosslinks (TOP-DPCs) coupled with DNA breaks. We discuss the expanding number of redundant pathways that repair TOP-DPCs, and the defects in those pathways, which are increasingly recognized as source of genomic damage leading to neurological diseases and cancer.

RIF1 acts in DNA repair through phosphopeptide recognition of 53BP1.

The chromatin-binding protein 53BP1 promotes DNA repair by orchestrating the recruitment of downstream effectors including PTIP, RIF1, and shieldin to DNA double-strand break sites. While we know how PTIP recognizes 53BP1, the molecular details of RIF1 recruitment to DNA-damage sites remains undefined. Here, we report that RIF1 is a phosphopeptide-binding protein that directly interacts with three phosphorylated 53BP1 epitopes. The RIF1-binding sites on 53BP1 share an essential LxL motif followed by two closely apposed phosphorylated residues. Simultaneous mutation of these sites on 53BP1 abrogates RIF1 accumulation into ionizing-radiation-induced foci, but surprisingly, only fully compromises 53BP1-dependent DNA repair when an alternative mode of shieldin recruitment to DNA-damage sites is also disabled. Intriguingly, this alternative mode of recruitment still depends on RIF1 but does not require its interaction with 53BP1. RIF1 therefore employs phosphopeptide recognition to promote DNA repair but also modifies shieldin action independently of 53BP1 binding.

Role of 53BP1 in end protection and DNA synthesis at DNA breaks.

Double-strand break (DSB) repair choice is greatly influenced by the initial processing of DNA ends. 53BP1 limits the formation of recombinogenic single-strand DNA (ssDNA) in BRCA1-deficient cells, leading to defects in homologous recombination (HR). However, the exact mechanisms by which 53BP1 inhibits DSB resection remain unclear. Previous studies have identified two potential pathways: protection against DNA2/EXO1 exonucleases presumably through the Shieldin (SHLD) complex binding to ssDNA, and localized DNA synthesis through the CTC1-STN1-TEN1 (CST) and DNA polymerase α (Polα) to counteract resection. Using a combinatorial approach of END-seq, SAR-seq, and RPA ChIP-seq, we directly assessed the extent of resection, DNA synthesis, and ssDNA, respectively, at restriction enzyme-induced DSBs. We show that, in the presence of 53BP1, Polα-dependent DNA synthesis reduces the fraction of resected DSBs and the resection lengths in G0/G1, supporting a previous model that fill-in synthesis can limit the extent of resection. However, in the absence of 53BP1, Polα activity is sustained on ssDNA yet does not substantially counter resection. In contrast, EXO1 nuclease activity is essential for hyperresection in the absence of 53BP1. Thus, Polα-mediated fill-in partially limits resection in the presence of 53BP1 but cannot counter extensive hyperresection due to the loss of 53BP1 exonuclease blockade. These data provide the first nucleotide mapping of DNA synthesis at resected DSBs and provide insight into the relationship between fill-in polymerases and resection exonucleases.

TP53 loss initiates chromosomal instability in fallopian tube epithelial cells.

High-grade serous ovarian cancer (HGSOC) originates in the fallopian tube epithelium and is characterized by ubiquitous TP53 mutation and extensive chromosomal instability (CIN). However, direct causes of CIN, such as mutations in DNA replication and mitosis genes, are rare in HGSOC. We therefore asked whether oncogenic mutations that are common in HGSOC can indirectly drive CIN in non-transformed human fallopian tube epithelial cells. To model homologous recombination deficient HGSOC, we sequentially mutated TP53 and BRCA1 then overexpressed MYC. Loss of p53 function alone was sufficient to drive the emergence of subclonal karyotype alterations. TP53 mutation also led to global gene expression changes, influencing modules involved in cell cycle commitment, DNA replication, G2/M checkpoint control and mitotic spindle function. Both transcriptional deregulation and karyotype diversity were exacerbated by loss of BRCA1 function, with whole-genome doubling events observed in independent p53/BRCA1-deficient lineages. Thus, our observations indicate that loss of the key tumour suppressor TP53 is sufficient to deregulate multiple cell cycle control networks and thereby initiate CIN in pre-malignant fallopian tube epithelial cells. This article has an associated First Person interview with the first author of the paper.

LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells.

DNA double-strand break (DSB) repair by homologous recombination (HR) is thought to be restricted to the S- and G2- phases of the cell cycle in part due to 53BP1 antagonizing DNA end resection in G1-phase and non-cycling quiescent (G0) cells. Here, we show that LIN37, a component of the DREAM transcriptional repressor, functions in a 53BP1-independent manner to prevent DNA end resection and HR in G0 cells. Loss of LIN37 leads to the expression of HR proteins, including BRCA1, BRCA2, PALB2, and RAD51, and promotes DNA end resection in G0 cells even in the presence of 53BP1. In contrast to 53BP1-deficiency, DNA end resection in LIN37-deficient G0 cells depends on BRCA1 and leads to RAD51 filament formation and HR. LIN37 is not required to protect DNA ends in cycling cells at G1-phase. Thus, LIN37 regulates a novel 53BP1-independent cell phase-specific DNA end protection pathway that functions uniquely in quiescent cells.

The WRN helicase: resolving a new target in microsatellite unstable cancers.

One of the goals of precision medicine is to uncover selective vulnerabilities in various cancers. A notable success has been the development of PARP inhibitors for the treatment of breast and ovarian cancers with mutations in BRCA genes. Only two years ago, it was discovered that cancers with microsatellite instability (MSI) were selectively dependent on the RecQ DNA helicase WRN. Subsequently, the molecular mechanism underlying WRN dependency in MSI cancers was uncovered. Here, we review how these developments have led to a promising new drug target in MSI cancers.

Two mechanisms of chromosome fragility at replication-termination sites in bacteria.

Chromosomal fragile sites are implicated in promoting genome instability, which drives cancers and neurological diseases. Yet, the causes and mechanisms of chromosome fragility remain speculative. Here, we identify three spontaneous fragile sites in the Escherichia coli genome and define their DNA damage and repair intermediates at high resolution. We find that all three sites, all in the region of replication termination, display recurrent four-way DNA or Holliday junctions (HJs) and recurrent DNA breaks. Homology-directed double-strand break repair generates the recurrent HJs at all of these sites; however, distinct mechanisms of DNA breakage are implicated: replication fork collapse at natural replication barriers and, unexpectedly, frequent shearing of unsegregated sister chromosomes at cell division. We propose that mechanisms such as both of these may occur ubiquitously, including in humans, and may constitute some of the earliest events that underlie somatic cell mosaicism, cancers, and other diseases of genome instability.

Mechanisms driving chromosomal translocations: lost in time and space.

Translocations arise when an end of one chromosome break is mistakenly joined to an end from a different chromosome break. Since translocations can lead to developmental disease and cancer, it is important to understand the mechanisms leading to these chromosome rearrangements. We review how characteristics of the sources and the cellular responses to chromosome breaks contribute to the accumulation of multiple chromosome breaks at the same moment in time. We also discuss the important role for chromosome break location; how translocation potential is impacted by the location of chromosome breaks both within chromatin and within the nucleus, as well as the effect of altered mobility of chromosome breaks. A common theme in work addressing both temporal and spatial contributions to translocation is that there is no shortage of examples of factors that promote translocation in one context, but have no impact or the opposite impact in another. Accordingly, a clear message for future work on translocation mechanism is that unlike normal DNA metabolic pathways, it isn't easily modeled as a simple, linear pathway that is uniformly followed regardless of differing cellular contexts.

The dystonia gene THAP1 controls DNA double-strand break repair choice.

The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.

END-seq: An Unbiased, High-Resolution, and Genome-Wide Approach to Map DNA Double-Strand Breaks and Resection in Human Cells.

DNA double-strand breaks (DSBs) represent the most toxic form of DNA damage and can arise in either physiological or pathological conditions. If left unrepaired, these DSBs can lead to genome instability which serves as a major driver to tumorigenesis and other pathologies. Consequently, localizing DSBs and understanding the dynamics of break formation and the repair process are of great interest for dissecting underlying mechanisms and in the development of targeted therapies. Here, we describe END-seq, a highly sensitive next-generation sequencing technique for quantitatively mapping DNA double-strand breaks (DSB) at nucleotide resolution across the genome in an unbiased manner. END-seq is based on the direct ligation of a sequencing adapter to the ends of DSBs and provides information about DNA processing (end resection) at DSBs, a critical determinant in the selection of repair pathways. The absence of cell fixation and the use of agarose for embedding cells and exonucleases for blunting the ends of DSBs are key advances that contribute to the technique's increased sensitivity and robustness over previously established methods. Overall, END-seq has provided a major technical advance for mapping DSBs and has also helped inform the biology of complex biological processes including genome organization, replication fork collapse and chromosome fragility, off-target identification of RAG recombinase and gene-editing nucleases, and DNA end resection at sites of DSBs.

Repeat expansions confer WRN dependence in microsatellite-unstable cancers.

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.