Molecular Analysis of the Replication Stress Response Caused by DDX11 Dysfunction and Overexpression

Jegadesan, Nanda Kumar (2022). Molecular Analysis of the Replication Stress Response Caused by DDX11 Dysfunction and Overexpression. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00013e9b

Abstract

DDX11 is a conserved iron-sulfur cluster DNA helicase, dysregulated in various cancers and important for normal development. While DDX11 plays roles in DNA repair and mitotic chromosome structure, the molecular mechanism by which DDX11 affects DNA damage response and tumorigenesis is still elusive. We report that loss/depletion of DDX11 in various cancer cell lines, but not in transformed normal cells, confers sensitivity to chemotherapeutic drugs, including Olaparib and Cisplatin. We established DDX11 knockout (KO) and performed synthetic lethality drug screens, uncovering that DDX11 loss sensitizes cells to ATR inhibitors, TOPII poisons and p53 stabilizers. DDX11 averts accumulation of double strand breaks in unperturbed and chemotherapeutic drug treated conditions and plays a crucial role in protecting against MRE11-mediated nascent strand degradation upon replication stress and fork stalling conditions.

Focusing on the mechanism by which DDX11 facilitates cellular survival upon DNA damage, we find that DDX11 promotes homologous recombination (HR)-mediated double strand break (DSB) repair by facilitating single stranded DNA (ssDNA) formation and subsequently RPA and RAD51 focus formation. By quantitively measuring ssDNA upon induction of DSBs at specific loci, we find that loss of DDX11 significantly impairs DNA end resection. DDX11 inactivation is synergistic to chemotherapeutic drugs in BRCA1/2-deficient cells by aggravating the DNA damage accumulation. Notably, loss of 53BP1 in DDX11 KO cells partially rescues the HR-defects and their sensitivity to Olaparib and Cisplatin. Mechanistically, DDX11 works downstream of the NHEJ factor 53BP1 to facilitate DNA end resection, RAD51 focus formation and to mediate HR-mediated DSB repair non-redundantly with the HR mediators BRCA1 and BRCA2. Importantly, downregulation of DDX11 re-sensitized BRCA1/2-mutated drug-resistant cancer cells that regained HR proficiency and drug resistance to PARP inhibitors and platinum drugs. Altogether, our results indicate DDX11 as a potential target for both BRCA1/2-deficient as well as HR proficient and drug resistant cancer cells.

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