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Koussa, N. C.

Publications and source records attributed to Koussa, N. C..

2 recordsLinked to original sources

Comprehensive mapping of cell fates in microsatellite unstable cancer cells support dual targeting of WRN and ATR

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN, knowledge that would be helpful for informing clinical development of WRN-targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system wherein the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We find that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we find no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provided the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggested a potential therapeutical rationale for dual targeting of WRN and ATR.

cancer biology↗

Reduced cellular levels of DNA polymerase delta alter replication dynamics and enzymology, and impair lagging-strand processing

DNA polymerase delta (Pol{delta} ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol{delta} is responsible for the synthesis and processing of the lagging-strand. At replication origins, Pol{delta} has been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Pol{delta} subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol{delta} in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze how the depletion of Pol{delta} impacts replication origin firing and lagging-strand synthesis during replication elongation in vivo in S. cerevisiae. By analyzing nascent lagging-strand products, we observe a genome-wide change in both the establishment and progression of replication. S-phase progression is slowed in Pol{delta} depletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Pol{delta} is capable of synthesizing a substantial amount of lagging-strand DNA, even when Pol{delta} is severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Pol{delta} is limiting; these defects lead to a strict dependence on checkpoint signaling and resection-mediated repair pathways for cellular viability. SIGNIFICANCE STATEMENTDNA replication in eukaryotes is carried out by the replisome - a multi-subunit complex comprising the enzymatic activities required to generate two intact daughter DNA strands. DNA polymerase delta (Pol{delta} ) is a multi-functional replisome enzyme responsible for synthesis and processing of the lagging-strand. Mutations in Pol{delta} cause a variety of human diseases: for example, destabilizing mutations lead to immunodeficiency. We titrate the concentration of Pol{delta} in budding yeast - a simple model eukaryote with conserved DNA replication machinery. We characterize several replication defects associated with Pol{delta} scarcity. The defects we observe provide insight into how destabilizing Pol{delta} mutations lead to genome instability.

molecular biology↗