An extended Tudor domain within Vreteno interconnects Gtsf1L and Ago3 for piRNA biogenesis in Bombyx mori.

Piwi-interacting RNAs (piRNAs) direct PIWI proteins to transposons to silence them, thereby preserving genome integrity and fertility. The piRNA population can be expanded in the ping-pong amplification loop. Within this process, piRNA-associated PIWI proteins (piRISC) enter a membraneless organelle called nuage to cleave their target RNA, which is stimulated by Gtsf proteins. The resulting cleavage product gets loaded into an empty PIWI protein to form a new piRISC complex. However, for piRNA amplification to occur, the new RNA substrates, Gtsf-piRISC, and empty PIWI proteins have to be in physical proximity. In this study, we show that in silkworm cells, the Gtsf1 homolog BmGtsf1L binds to piRNA-loaded BmAgo3 and localizes to granules positive for BmAgo3 and BmVreteno. Biochemical assays further revealed that conserved residues within the unstructured tail of BmGtsf1L directly interact with BmVreteno. Using a combination of AlphaFold modeling, atomistic molecular dynamics simulations, and in vitro assays, we identified a novel binding interface on the BmVreteno-eTudor domain, which is required for BmGtsf1L binding. Our study reveals that a single eTudor domain within BmVreteno provides two binding interfaces and thereby interconnects piRNA-loaded BmAgo3 and BmGtsf1L.

Spatial Chromosome Folding and Active Transcription Drive DNA Fragility and Formation of Oncogenic MLL Translocations.

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 CCCTC-binding factor (CTCF) and 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 hotspots 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.

PARP1 proximity proteomics reveals interaction partners at stressed replication forks.

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.

53BP1-DNA repair enters a new liquid phase.

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.

Studies of the DNA Damage Response by Using the Lac Operator/Repressor (LacO/LacR) Tethering System.

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.