Somatic instability of the FGF14 -SCA27B GAA•TTC repeat reveals a marked expansion bias in the cerebellum.

Spinocerebellar ataxia 27B (SCA27B) is a common autosomal dominant ataxia caused by an intronic GAA•TTC repeat expansion in FGF14 . Neuropathological studies have shown that neuronal loss is largely restricted to the cerebellum. Although the repeat locus is highly unstable during intergenerational transmission, it remains unknown whether it exhibits cerebral mosaicism and progressive instability throughout life. We conducted an analysis of the FGF14 GAA•TTC repeat somatic instability across 156 serial blood samples from 69 individuals, fibroblasts, induced pluripotent stem cells, and post-mortem brain tissues from six controls and six patients with SCA27B, alongside methylation profiling using targeted long-read sequencing. Peripheral tissues exhibited minimal somatic instability, which did not significantly change over periods of more than 20 years. In post-mortem brains, the GAA•TTC repeat was remarkably stable across all regions, except in the cerebellar hemispheres and vermis. The levels of somatic expansion in the cerebellar hemispheres and vermis were, on average, 3.15 and 2.72 times greater relative to other examined brain regions, respectively. Additionally, levels of somatic expansion in the brain increased with repeat length and tissue expression of FGF14 . We found no significant difference in methylation of wild-type and expanded FGF14 alleles in post-mortem cerebellar hemispheres between patients and controls. In conclusion, our study revealed that the FGF14 GAA•TTC repeat exhibits a cerebellar-specific expansion bias, which may explain the pure and late-onset cerebellar involvement in SCA27B.

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing.

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3'-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes.

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

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 pharmacological 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 a role for SOD1 in the survival of PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.

A common flanking variant is associated with enhanced stability of the FGF14-SCA27B repeat locus.

The factors driving or preventing pathological expansion of tandem repeats remain largely unknown. Here, we assessed the FGF14 (GAA)·(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a common 5'-flanking variant in 70.34% of alleles analyzed (3,463/4,923) that represents the phylogenetically ancestral allele and is present on all major haplotypes. This common sequence variation is present nearly exclusively on nonpathogenic alleles with fewer than 30 GAA-pure triplets and is associated with enhanced stability of the repeat locus upon intergenerational transmission and increased Fiber-seq chromatin accessibility.

Structure and repair of replication-coupled DNA breaks.

Using CRISPR/Cas9 nicking enzymes, we examine the interaction between the replication machinery and single strand breaks, one of the most common forms of endogenous DNA damage. We show that replication fork collapse at leading strand nicks generates resected single-ended double-strand breaks (seDSBs) that are repaired by homologous recombination (HR). If these seDSBs are not promptly repaired, arrival of adjacent forks creates double ended DSBs (deDSBs), which could drive genomic scarring in HR-deficient cancers. deDSBs can also be generated directly when the replication fork bypasses lagging strand nicks. Unlike deDSBs produced independently of replication, end-resection at nick-induced se/deDSBs is BRCA1-independent. Nevertheless, BRCA1 antagonizes 53BP1 suppression of RAD51 filament formation. These results highlight unique mechanisms that maintain replication fork stability.

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.

Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures.

CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (A2G3)n repeat in the RFC1 gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the pathogenic (A2G3)n and nonpathogenic (A4G)n repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization in vitro. The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in vitro reveals triplex H-DNA formation by only the pathogenic repeat. Consistently, bioinformatic analysis of S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A2G3)n motifs over (A4G)n motifs. Finally, the pathogenic, but not the nonpathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that the CANVAS-causing (A2G3)n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication.

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.

Detection of alternative DNA structures and its implications for human disease.

Around 3% of the genome consists of simple DNA repeats that are prone to forming alternative (non-B) DNA structures, such as hairpins, cruciforms, triplexes (H-DNA), four-stranded guanine quadruplexes (G4-DNA), and others, as well as composite RNA:DNA structures (e.g., R-loops, G-loops, and H-loops). These DNA structures are dynamic and favored by the unwinding of duplex DNA. For many years, the association of alternative DNA structures with genome function was limited by the lack of methods to detect them in vivo. Here, we review the recent advancements in the field and present state-of-the-art technologies and methods to study alternative DNA structures. We discuss the limitations of these methods as well as how they are beginning to provide insights into causal relationships between alternative DNA structures, genome function and stability, and human disease.

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.

Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures.

CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (A2G3)n repeat in the RFC1 gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the main pathogenic (A2G3)n and the main nonpathogenic (A4G)n repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization in vitro. The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in supercoiled DNA reveals triplex H-DNA formation by the pathogenic repeat. Consistently, bioinformatic analysis of the S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A2G3)n motifs over (A4G)n motifs in vivo. Finally, the pathogenic, but not the non-pathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that CANVAS-causing (A2G3)n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication.

The mission to ensure continued funding for excellent basic research.

The surprising decision by Novo Nordisk Foundation (NNF) to discontinue funding for the Center for Protein Research in Copenhagen should prompt discussions about public and private commitment to support basic research.

Rapid and precise genotyping of transgene zygosity in mice using an allele-specific method.

Precise determination of transgene zygosity is essential for use of transgenic mice in research. Because integration loci of transgenes are usually unknown due to their random insertion, assessment of transgene zygosity remains a challenge. Current zygosity genotyping methods (progeny testing, qPCR, and NGS-computational biology analysis) are time consuming, prone to error or technically challenging. Here, we developed a novel method to determine transgene zygosity requiring no knowledge of transgene insertion loci. This method applies allele-specific restriction enzyme digestion of PCR products (RE/PCR) to rapidly and reliably quantify transgene zygosity. We demonstrate the applicability of this method to three transgenic strains of mice (Atm TgC3001L, Nes-Cre, and Syn1-Cre) harboring a unique restriction enzyme site on either the transgene or its homologous sequence in the mouse genome. This method is as accurate as the gold standard of progeny testing but requires 2 d instead of a month or more. It is also exceedingly more accurate than the most commonly used approach of qPCR quantification. Our novel method represents a significant technical advance in determining transgene zygosities in mice.

Centromeres as universal hotspots of DNA breakage, driving RAD51-mediated recombination during quiescence.

Centromeres are essential for chromosome segregation in most animals and plants yet are among the most rapidly evolving genome elements. The mechanisms underlying this paradoxical phenomenon remain enigmatic. Here, we report that human centromeres innately harbor a striking enrichment of DNA breaks within functionally active centromere regions. Establishing a single-cell imaging strategy that enables comparative assessment of DNA breaks at repetitive regions, we show that centromeric DNA breaks are induced not only during active cellular proliferation but also de novo during quiescence. Markedly, centromere DNA breaks in quiescent cells are resolved enzymatically by the evolutionarily conserved RAD51 recombinase, which in turn safeguards the specification of functional centromeres. This study highlights the innate fragility of centromeres, which may have been co-opted over time to reinforce centromere specification while driving rapid evolution. The findings also provide insights into how fragile centromeres are likely to contribute to human disease.

Multiscale reorganization of the genome following DNA damage facilitates chromosome translocations via nuclear actin polymerization.

Nuclear actin-based movements have been shown to orchestrate clustering of DNA double-strand breaks (DSBs) into homology-directed repair domains. Here we describe multiscale three-dimensional genome reorganization following DNA damage and analyze the contribution of the nuclear WASP-ARP2/3-actin pathway toward chromatin topology alterations and pathologic repair. Hi-C analysis reveals genome-wide, DNA damage-induced chromatin compartment flips facilitated by ARP2/3 that enrich for open, A compartments. Damage promotes interactions between DSBs, which in turn facilitate aberrant, actin-dependent intra- and inter-chromosomal rearrangements. Our work establishes that clustering of resected DSBs into repair domains by nuclear actin assembly is coordinated with multiscale alterations in genome architecture that enable homology-directed repair while also increasing nonhomologous end-joining-dependent translocation frequency.

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.