Isolation and detection of DNA-protein crosslinks in mammalian cells.

DNA-protein crosslinks (DPCs) are toxic DNA lesions wherein a protein is covalently attached to DNA. If not rapidly repaired, DPCs create obstacles that disturb DNA replication, transcription and DNA damage repair, ultimately leading to genome instability. The persistence of DPCs is associated with premature ageing, cancer and neurodegeneration. In mammalian cells, the repair of DPCs mainly relies on the proteolytic activities of SPRTN and the 26S proteasome, complemented by other enzymes including TDP1/2 and the MRN complex, and many of the activities involved are essential, restricting genetic approaches. For many years, the study of DPC repair in mammalian cells was hindered by the lack of standardised assays, most notably assays that reliably quantified the proteins or proteolytic fragments covalently bound to DNA. Recent interest in the field has spurred the development of several biochemical methods for DPC analysis. Here, we critically analyse the latest techniques for DPC isolation and the benefits and drawbacks of each. We aim to assist researchers in selecting the most suitable isolation method for their experimental requirements and questions, and to facilitate the comparison of results across different laboratories using different approaches.

The NUCKS1-SKP2-p21/p27 axis controls S phase entry.

Efficient entry into S phase of the cell cycle is necessary for embryonic development and tissue homoeostasis. However, unscheduled S phase entry triggers DNA damage and promotes oncogenesis, underlining the requirement for strict control. Here, we identify the NUCKS1-SKP2-p21/p27 axis as a checkpoint pathway for the G1/S transition. In response to mitogenic stimulation, NUCKS1, a transcription factor, is recruited to chromatin to activate expression of SKP2, the F-box component of the SCFSKP2 ubiquitin ligase, leading to degradation of p21 and p27 and promoting progression into S phase. In contrast, DNA damage induces p53-dependent transcriptional repression of NUCKS1, leading to SKP2 downregulation, p21/p27 upregulation, and cell cycle arrest. We propose that the NUCKS1-SKP2-p21/p27 axis integrates mitogenic and DNA damage signalling to control S phase entry. The Cancer Genome Atlas (TCGA) data reveal that this mechanism is hijacked in many cancers, potentially allowing cancer cells to sustain uncontrolled proliferation.

AsiDNA Treatment Induces Cumulative Antitumor Efficacy with a Low Probability of Acquired Resistance.

The Achilles heel of anticancer treatments is intrinsic or acquired resistance. Among many targeted therapies, the DNA repair inhibitors show limited efficacy due to rapid emergence of resistance. We examined evolution of cancer cells and tumors treated with AsiDNA, a new DNA repair inhibitor targeting all DNA break repair pathways. Effects of AsiDNA or Olaparib were analyzed in various cell lines. Frequency of AsiDNA- and olaparib-resistant clones was measured after 2 weeks of continuous treatment in KBM7 haploid cells. Cell survivals were also measured after one to six cycles of 1-week treatment and 1-week recovery in MDA-MB-231 and NCI-H446. Transcriptomes of cell populations recovering from cyclic treatments or mock treatment were compared. MDA-MB-231 xenografted models were treated with three cycles of AsiDNA to monitor the effects of treatment on tumor growth and transcriptional modifications. No resistant clones were selected after AsiDNA treatment (frequency < 3x10-8) in treatment conditions that generate resistance to olaparib at a frequency of 7.2x10-7 resistant clones per treated cell. Cyclic treatments promote cumulative sensitivity characterized by a higher mortality of cells having undergone previous treatment cycles. This sensitization was stable, and transcriptome analysis revealed a major gene downregulation with a specific overrepresentation of genes coding for targets of DNA-PK. Such changes were also detected in tumor models which showed impaired growth after cycles of AsiDNA treatment.