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.

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.

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.

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.

Neuronal enhancers are hotspots for DNA single-strand break repair.

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.

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.