DNA damage is a hallmark of cancer, and mutation and misregulation of proteins that maintain genomic fidelity are associated with the development of multiple cancers. DNA double strand breaks are arguably considered the most deleterious type of DNA damage. The nonhomologous end-joining (NHEJ) pathway is one mechanism to repair DNA double strand breaks, and proteins involved in NHEJ may also regulate DNA replication. We previously established that DNA-PKcs, a NHEJ protein, promotes genomic stability and cell viability following cellular exposure to replication stress; we wanted to discern whether another NHEJ protein, DNA ligase IV (Lig4), shares this phenotype. Our investigations focused on triple negative breast cancer cells, as, compared to nonbasal breast cancer, LIG4 is frequently amplified, and an increased gene dose is associated with higher Lig4 expression. We depleted Lig4 using siRNA and confirmed our knockdown by qPCR and western blotting. Cell survival diminished with Lig4 depletion alone, and this was associated with increased replication fork stalling. Checkpoint protein Chk1 activation and dephosphorylation were unchanged in Lig4-depleted cells. Lig4 depletion resulted in sustained DNA-PKcs phosphorylation following hydroxyurea exposure. Understanding the effect of Lig4 on genomic replication and the replication stress response will clarify the biological ramifications of inhibiting Lig4 activity. In addition, Lig4 is an attractive clinical target for directing CRISPR/Cas9-mediated repair towards homology-directed repair and away from NHEJ, thus understanding of how diminishing Lig4 impacts cell biology is critical.
Maintenance of the genome is essential for cell survival, and impairment of the DNA damage response is associated with multiple pathologies including cancer and neurological abnormalities. DNA-PKcs is a DNA repair protein and a core component of the classical nonhomologous end-joining pathway, but it may have roles in modulating gene expression and thus, the overall cellular response to DNA damage. Using cells producing either wild-type (WT) or kinase-inactive (KR) DNA-PKcs, we assessed global alterations in gene expression in the absence or presence of DNA damage. We evaluated differential gene expression in untreated cells and observed differences in genes associated with cellular adhesion, cell cycle regulation, and inflammation-related pathways. Following exposure to etoposide, we compared how KR versus WT cells responded transcriptionally to DNA damage. Downregulation of pathways involved in biosynthesis were observed in both genotypes, but upregulated biological pathways were divergent, again with KR cells manifesting a more robust inflammatory response compared to WT cells. To determine what major transcriptional regulators are controlling the differences in gene expression noted, we used pathway analysis and found that many master regulators of histone modifications, proinflammatory pathways, cell cycle regulation, Wnt/β-catenin signaling, and cellular development and differentiation were impacted by DNA-PKcs status. Overall, our results indicate that DNA-PKcs, in a kinase-dependent fashion, decreases proinflammatory signaling following genotoxic insult. As multiple DNA-PK kinase inhibitors are in clinical trial as cancer therapeutics utilized in combination with DNA damaging agents, understanding the transcriptional response when DNA-PKcs cannot phosphorylate downstream targets will inform the overall patient response to combined treatment.
R‐loops are three stranded nucleic acid structures that form during transcription; when the nascent strand of RNA invades the replication bubble, hybridizes with the DNA template strand and displaces the non‐template strand. For many years, these DNA:RNA hybrids were thought to only be byproducts of transcription. In recent years, however, it has been shown that R‐loops play many roles in diverse cellular processes, such as immunoglobulin class‐switching recombination, epigenetic regulation, and in DNA replication. R‐loop mis regulation can lead to DNA damage and genomic instability. Additionally, R‐loops have been shown to play a role in several neurodegenerative diseases. Although, our knowledge of R‐loops has grown in the last few years, detection and quantification of R‐loops can still be difficult and inaccurate. Here, we provide a novel method for quantification of nuclear R‐loops based on cellular fractionation and compare it with commonly used R‐loop quantification methods.Support or Funding InformationResearch was funded by HHMI Research Scholars program at NMSU, Cowboys for Cancer Research and the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103451.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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