Decoy Damaged DNA Discovers New Gene Repair Protein

Researchers used a synthetic DNA structure to mimic an intermediate of homologous recombination, the most reliable cell cycle process to repair DNA correctly. This DNA structure was then used as bait to capture nuclear proteins in the hopes of identifying a new player in the cellular response to DNA damage. These proteins were isolated and subsequently identified through mass spectrometry, revealing that the Heterogeneous Nuclear Ribonucleoprotein D (HNRNPD) was indeed able to bind chromatin DNA, a prerequisite for a protein involved in DNA repair, and to re-localize specifically onto DNA damaged sites.

The study reporting on this newly discovered role for HNRNPD, previously known for its role in messenger RNA regulation, was recently published in Nucleic Acids Research, one of the most authoritative journals in the field, from the Oxford Academic Press, by the research team lead by Antonio Giordano, M.D., Ph.D., Director of the Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, and Professor of Pathology, University of Siena, Italy.

Because protecting the genome against DNA damage is crucial to prevent harmful mutations and cancer development, the authors utilized a ‘gene fishing’ approach using the synthetic DNA structure that was designed by Luigi Alfano, a postdoctoral fellow at the National Cancer Institute of Naples, Pascale Foundation-CROM in Mercogliano, working in the Cell Cycle & Cancer Lab coordinated by Francesca Pentimalli, a longtime collaborator of Prof. Giordano and Adjunct Professor at the Sbarro Institute. The captured proteins were analyzed under mass spectrometry by Luca Bini and Claudia Landi at the University of Siena. Alfano and colleagues focused on the RNA-binding protein HNRNPD, the loss of which induces cell senescence and premature aging in mice, two features associated with a defective DNA damage response.

Upon DNA damage, cells activate homologous recombination repair to cut the DNA near the break (a process known as DNA end resection), generating a single stranded DNA tail that is able to find the complementary homologous sequence within the sister chromatid, using it as a template for faithful repair. The authors found that silencing HNRNPD expression impaired the DNA end resection process, affecting the overall DNA damage response. Similarly, depleting HNRNPD through CRISPR/Cas9-mediated gene editing, impaired the cell response to DNA damage induced by the chemotherapy drug camptothecin, making cancer cells more susceptible to this drug and also to olaparib, a drug that targets specifically the DNA repair process used against some types of breast and ovarian cancer.

“The inhibition of HNRNPD, through chemical compounds, can be used as a new strategy for cancer treatment in a combination therapy with the PARP1 inhibitor (Olaparib),” Giordano says. “Based on the concept of synthetic lethality, this potential clinical application is analogous to the situation described for the BRCA cancers.”

Delving deeper into the underlying molecular mechanisms, the authors found that HNRNPD interacts with SAF-A, another RNA-binding protein previously found correlated to the DNA damage response. The authors showed that HNRNPD silencing impaired the loading of SAF-A onto chromatin upon DNA damage. Moreover, HNRNPD silencing caused an accumulation, onto damaged DNA, of DNA:RNA hybrids (also called R-loops) whose proper removal is required to preserve genome integrity. Indeed, expressing RNase H, an enzyme that digests the RNA within the hybrids, or inhibiting RNA formation through alpha-amanitin, could rescue the phenotype of HNRNPD knockout cells, reinstating an effective DNA damage response.

“Overall, our data strengthens the role of RNA-binding proteins in the DNA repair mechanism and identify HNRNPD as a new key player in DNA repair,”  says lead author Alfano. “They also provide new clues on the still poorly defined function of R-loop role in DNA damage repair.”

“Targeting DNA repair pathways proved to be a powerful approach for cancer therapy, as epitomized by the clinical use of olaparib for various tumors,” say co-authors Pentimalli and Giordano. “The identification of HNRNPD as an homologous recombination protein could be useful to design new synthetic lethal approaches and also inform genome editing strategies that use endogenous cell repair pathways to modify DNA sequences.”

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