Elucidating the role of NEK8 in the replication stress response [electronic resource]

The genome is under constant threat by DNA damage arising from endogenous and exogenous sources, and it is particularly susceptible to damage during DNA replication. Using a genome-wide siRNA screen to identify proteins necessary for genome maintenance in the presence of replication stress, we identified NEK8/NPHP9, a ciliary protein kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease (PKD). Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. Recent findings have shown that several genes linked to the DNA damage signaling are mutated in patients with NPHP or NPHP-like diseases. However, whether loss of the DNA damage response (DDR) itself played a causal role in NPHP is not clear, and the underlying mechanism linking these genes to NPHP is not addressed. This thesis work provides new mechanistic insight into the functions of the NIMA-related kinase, NEK8, in suppressing DNA damage arising from replication stress, and for the first time connects the replication stress response to cystic kidney diseases and renal ciliopathies. Here we directly demonstrate that replication stress, caused by the DNA polymerase inhibitor aphidicolin, as well as NEK8 loss, affects renal cell architecture in a 3D-culture system. Moreover, NEK8 mutation leads to replication stress and DNA damage in the pre-cystic kidney. We further establish that NEK8 is a crucial effector of the replication stress response and that it functions as part of the ATR-dependent signaling pathway, regulating replication fork dynamics and fork stability through effects on CDK activity. We also show NEK8's role in the replication stress response is compromised by ciliopathy-causing mutations. Thus, our study strongly suggests that excessive replication stress or a defect in the ability of cells to respond to replication stress leads to chronic damage in renal epithelial cells and kidney failure in ciliopathies. The major contributions of this dissertation are: (1) We validated NEK8 as a high confidence hit from a previously described unbiased siRNA screen designed to uncover genes that help maintain genome stability during DNA replication stress. (2) Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) which further accumulate when replication forks stall. (3) NEK8 acts at the replication fork to regulate fork dynamics and replication origin firing, particularly under conditions of replication stress. (4) NEK8 functions in the ATR pathway, interacting with ATR and other components of the replication checkpoint, and it suppresses S-phase CDK activity during replication to prevent premature origin firing. (5) NEK8 mutations, which produce kinase-inactive protein or juvenile cystic kidneys (jck), abolish the interaction of NEK8 with ATR and fail to prevent DNA damage accumulation in NEK8-deficient cells. (6) Disruption of NEK8, as well as replication stress induced by aphidicolin, perturbs both ciliogenesis and spheroid structure in a 3D assay that models renal kidney architecture and NPHP. This study therefore provides the first direct evidence that replication stress contributes to renal ciliopathies and mechanistic insight into how it does so. Furthermore, it identifies a new critical component of the replication stress response: NEK8.

Keywords: Chemical and Systems Biology.
Advisor: Cimprich Karlene primary advisor. Thesis advisor
Elias Joshua advisor. Thesis advisor
Ferrell James Ellsworth advisor. Thesis advisor
Meyer Tobias advisor. Thesis advisor
Stearns Tim advisor. Thesis advisor