Highlights d TOP2-mediated DNA fragility is linked to transcription and proximity to loop anchors d Loop-anchor DNA fragility correlates with transcriptional activity and directionality d Genes forming oncogenic fusions are highly transcribed and enriched at loop anchors d Formation of MLL fusions relies on the activities of both TOP2 isoforms
How spatial chromosome organization influences genome integrity is still poorly understood.Here we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities, are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CTCF/cohesin bound sites at the bases of chromatin loops and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias, are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hot spots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability, and are key contributors to the occurrence of genome rearrangements that drive cancer. Reimand et al., 2016) and topological constraints during loop extrusion dynamics (Canela et al., 2017).Although physiologically important, TOP2 functions are also inherently risky for a cell, as they involve the formation of a transient DSB to allow the passage of duplex DNA through the break. During this controlled process, the two-TOP2 subunits are covalently linked through their active sites to each 5′-terminus of a DSB via a phosphodiester bond (Pommier et al., 2016).These key intermediates of TOP2 activity, called TOP2 cleavage complexes (TOP2ccs) are normally short-lived, as topoisomerases quickly ligate the DNA ends upon the passage of the duplex DNA. In the presence of nearby DNA lesions or upon treatment with topoisomerase poisons, however, these cleavage complexes can be stabilized on DNA and may contribute to genomic instability by acting as road blocks to DNA-tracking systems that attempt to traverse them (Ashour et al., 2015).The mechanism by which TOP2ccs are converted to DSBs underlies the action of widely used class of anticancer agents that trap TOP2 at the intermediate cleavage complex step (Nitiss, 2009). The TOP2 poison etoposide (ETO), is among the most effective and widely used anticancer agents in the clinic, but treatment with etoposide is associated with the occurrence of therapy-related acute myeloid leukemias (t-AMLs) (Allan and Travis, 2005; Wright and Vaughan, 2014). Approximately one third of t-AML cases are associated with recurrent chromosome translocations between the mixed lineage leukemia gene (MLL) and various potential translocation partners, and amongst them, AF9, AF4, AF6, and ENL, account for approximately 80% of the cases. Sequencing of MLL fusions from patients with t-AML has shown that MLL breakpoints occur in breakpoint cluster regions (BCRs) at restricted genomic positions near exon 12 of the MLL gene, while BCRs within potential translocation partners are ENL fusions two times more frequently than MLL-AF4, MLL-AF6, MLL-AF9 ( Figure 2D), although the frequency of ENL breakage was not signi...
In response to DNA damage, transient repair compartments in the nucleus concentrate repair proteins and activate downstream signaling factors. In this issue of The EMBO Journal, Kilic et al show that DNA repair focal assemblies marked by accumulation of 53BP1 are phase separated liquid compartments. This liquid droplet‐like behavior of 53BP1 compartments might help to coordinate local lesion recognition with global gene activation in response to DNA damage.
PARP1 mediates poly-ADP-ribosylation of proteins on chromatin in response to different types of DNA lesions. PARP inhibitors are used for the treatment of BRCA1/2-deficient breast, ovarian, and prostate cancer. Loss of DNA replication fork protection is proposed as one mechanism that contributes to the vulnerability of BRCA1/2-deficient cells to PARP inhibitors. However, the mechanisms that regulate PARP1 activity at stressed replication forks remain poorly understood. Here, we performed proximity proteomics of PARP1 and isolation of proteins on stressed replication forks to map putative PARP1 regulators. We identified TPX2 as a direct PARP1-binding protein that regulates the auto-ADP-ribosylation activity of PARP1. TPX2 interacts with DNA damage response proteins and promotes homology-directed repair of DNA double-strand breaks. Moreover, TPX2 mRNA levels are increased in BRCA1/2-mutated breast and prostate cancers, and high TPX2 expression levels correlate with the sensitivity of cancer cells to PARP-trapping inhibitors. We propose that TPX2 confers a mitosis-independent function in the cellular response to replication stress by interacting with PARP1.
Maintaining the integrity of genetic information is essential for the survival of cells. Recent advances in cell biological and microscopy methodologies have complemented traditional genetic and biochemical approaches, and they now permit the observation of spatiotemporal aspects of damaged chromosomal loci. In one of these approaches, integrated LacO/TetO operator sequences can be used as binding sites to physically tether onto chromatin any protein of interest when genetically fused to the respective repressors (LacR/TetR). This methodology has been the basis of several models to probe the spatial dynamics of DNA repair in the eukaryotic nucleus and to visualize genomic loci in yeast, fly, nematodes, and in mammalian cells. Further applications are the induction of localized DNA damage by immobilizing endonucleases at different genome sites in vivo, the assessment of the hierarchy of protein interactions within repair complexes, and the activation of the DNA damage response (DDR) by the physical tethering of DSB-repair factors on chromatin in the absence of damage. We outline here a protocol for the quantification of DDR activation upon the prolonged immobilization of single repair factors on chromatin or upon tethering of the endonuclease FokI. The outlined protocol requires basic cell culture and microscopy skills and allows the tethering of any protein of interest within 2-3 days.
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