Biology subjects

Melendy, T.

Publications and source records attributed to Melendy, T..

2 recordsLinked to original sources

bioRxiv2023

DNA damaged-induced phosphorylation of a viral replicative DNA helicase results in inhibition of DNA replication through attenuation of helicase function

A major function of the DNA damage responses (DDRs) that act during the replicative phase of the cell cycle is to inhibit initiation and elongation of DNA replication. The polyomavirus SV40 is an important model system for studying human DNA replication and DDRs due to its heavy reliance on host factors for viral DNA replication, and the arrest of SV40 DNA replication in response to DDR activation. The inhibition of SV40 DNA replication following DDR activation is associated with enhanced DDR kinase phosphorylation of SV40 Large T-antigen (LT), the viral origin-binding protein and DNA helicase. NetPhos prediction of LT phosphorylation on multiple sites were confirmed by mass spectroscopy, including a highly conserved DDR kinase site, T518. In cell-based DNA replication assays expression of the phosphomimetic mutant form of LT at T518 (T518D) resulted in dramatically decreased levels of SV40 DNA replication; while LT-dependent transcriptional activation was unaffected. WT and LT T518D were subsequently expressed, purified, and analyzed in vitro for assessment of biochemical function. In concordance with the cell-based data, reactions using SV40 LT-T518D, but not T518A, showed dramatic inhibition of SV40 DNA replication. Importantly, the LT T518D mutation did not affect critical LT protein interactions or its ATPase function, but showed decreased helicase activity on long, but not very short, DNA templates. These results suggest that DDR phosphorylation at T518 inhibits SV40 DNA replication by impeding LT helicase activity, thereby slowing the DNA replication fork. This is consistent with the slowing of cellular replication forks following DDR and may provide a paradigm for another mechanism for how DNA replication forks can be slowed in response to DDR, by phosphorylation of DNA helicases.

microbiology↗

bioRxiv2021

DDK inhibition disrupts replication leading to mitotic catastrophe in Ewing sarcoma

Ewing sarcoma is the second most common bone malignancy in children and adolescents. Patients with upfront metastatic or recurrent disease have poor outcomes with 5-year survival rates of <30%. CDC7, also known as DDK (DBF4-dependent kinase), is a serine-threonine kinase that, in coordination with its activation subunit ASK (or DBF4), is involved in a diverse array of cellular functions including the regulation of DNA replication initiation and activation of the replication stress response. Due to DDKs diverse roles during replication, coupled with an increased level of genomic instability and R-loop-mediated replication stress within Ewing sarcoma cells, we hypothesized that Ewing sarcoma cells would be particularly vulnerable to DDK inhibitors. Here, we show that treatment with two selective DDK inhibitors, TAK-931 and XL413, results in apoptosis and a significant reduction in cell viability in EWS-FLI1-harboring Ewing sarcoma cell lines. We show that low dose DDK inhibition in Ewing sarcoma cells causes an accumulation of cells in late-S phase with a reduced replication capacity. There is also evidence of premature mitotic entry indicating an inability to properly complete DNA replication in a timely manner upon DDK inhibition. Also, there is a significant increase in the formation of micronuclei and other aberrant mitotic structures upon DDK inhibition in Ewing sarcoma cells indicating a failure to properly progress through S-phase followed by improper mitotic entry/progression, resulting in mitotic catastrophe. Interestingly, we observed minimal signs of mitotic accumulation, despite clear evidence of replication and mitotic stress, suggesting a failure to properly enforce the mitotic checkpoint. Together, these results suggest that Ewing sarcoma cells rely on the activity of DDK to maintain cell viability and suggest that DDK inhibition may prove to be a viable therapeutic strategy for patients with Ewing sarcoma.

cancer biology↗