DNA damage can occur from extrinsic sources e.g., irradiation or intrinsic sources, e.g., unresolved transcriptional R-loops (DNA-RNA hybrids plus displaced single-stranded DNA). The cell employs multiple mechanisms not only to repair the damage but to also promote cell survival while repair is underway. One recently described mechanism promoting cell survival following DNA damage involves fragmentation and dispersal of Golgi-complex throughout the cytosol, aims known as Golgi-dispersal response (GDR) (Farber-Katz et al., 2014). Here, impaired GDR resulted in cell death. Because lymphocytes from patients with Wiskott-Aldrich syndrome (WAS), an X-linked primary immunodeficiency disorder, undergo spontaneous, accelerated apoptosis (Rawlings et al., 1999; Zhou et al., 2017), we hypothesized that impaired GDR underlies progressive lymphopenia in WAS, and that a defect in GDR contributes, at least in part, to the development of immunodeficiency. The concept that WASp has an essential cytoplasmic role in the DNA damage response and repair (DDR) signaling is innovative, because WASp role in cytoplasm has traditionally been restricted to remodeling F-actin cortical cytoskeleton in the context of TCR activation. In strong support of this hypothesis, are our preliminary findings demonstrating a profoundly inhibited GDR following ?-irradiation-induced DNA strand breaks in WASp-deficient T cells. That the GDR event could be of immediate physiological relevance to the lymphocyte biology is further supported by our findings that WASp-deficiency triggers ectopic accumulation of unresolved R loops, the latter, we show generates DNA double strand breaks (DSBs) during Th1 gene activation. We, therefore, propose that the transcription-linked DNA damage in WAS lymphocytes will not accompany cell protective GDR, and the degree of GDR dysfunction will correlate with disease severity grades in WAS.
Aim 1 focuses on elucidating the molecular mechanisms by which loss of WASp disrupts the GDR signaling pathway, focusing on the formation and function of DNA-PK- GOLPH3-F-actin-MYO18A complex during DNA damage, both ?-irradiation-mediated and transcriptional R-loop-mediated.
In Aim 2, we will ask how disease-causing, WAS gene mutations differ in disrupting GDR signaling in Th1, Th2, and B cells, and investigate how WAS mutations that cause increased F-actin or decreased F-actin differentially influence GDR competency. Therefore, if successful, we would identify a novel cytoplasmic role of WASp in the DDR pathway, and uncover GDR defect as etiologic of a primary immunodeficiency disorder.
Wiskott-Aldrich syndrome (WAS) is a genetic disease affecting boys, who suffer life-threatening infections and cancer. We have uncovered a new function of WASp in a Golgi response to DNA damage. Deeper understanding this novel role will better define the etiology of WAS.