Investigating the Role of Ubiquitin Signalling in the DNA Replication Stress Response.

DNA replication is constantly challenged by obstacles that impede fork progression, leading to replication stress (RS). If unresolved, RS contributes to genomic instability, which is a hallmark of cancer. To counteract RS, cells activate protective mechanisms, such as fork reversal, to enable replication restart. Reversed stalled forks are resected by nucleases if they are not protected by the RAD51 recombinase. Replication fork stability is important for cancer therapy because it confers chemoresistance in certain cancers.  

A source of RS is R-loop-associated replication stalling. R-loops occur during transcription when nascent RNA hybridizes to the template DNA forming RNA:DNA hybrids with the non-template DNA displaced as a single strand. R-loops support key cellular processes, but if unperturbed they hinder replication fork progression and induce RS. While R-loops have been associated with double-strand break repair, their precise role in the RS response and potential therapeutic implications remain unexplored. Understanding this mechanism could open new avenues for targeting cancer cells with high RS. 

In our lab, we have identified an E3 ubiquitin ligase (referred to here as UbLX) as a novel R-loop-proximal protein that is involved in the RS response. Importantly, loss of UbLX reduces R-loop levels, increases sensitivity to RS-inducing agents, and causes RS presumably through regulation of fork reversal. In addition, loss of UbLX causes replication fork resection. 

 Based on the above, we hypothesize that UbLX cooperates with R-loops to maintain replication fork stability. 

Objectives 

This project will:  

• Investigate how UbLX controls R-loops. 
• Unravel how R-loops and UbLX regulate the RS response. 
• Interrogate the role of R-loops and UbLX in cancer cells. 

This project aims to unravel a previously unidentified role of ubiquitin ligase UbLX and R-loops at the RS response that hold the potential to be utilised in targeted cancer therapies. 

To address these key questions, this project will employ a range of molecular and cell biology approaches. These techniques include mammalian cell cultures, CRISPR/CAS9 gene editing, DNA fibre assay, DRIP-QPCR, immunofluorescence, western blotting, iPOND and sensitivity assays. The successful candidate will join a dynamic and collaborative research environment within the Lancaster University Department of Biomedical and Life Sciences, with access to cutting-edge facilities and expertise in genome instability and cancer biology. 

Person specification 

Applicants should have a strong background in cell and molecular biology, and ideally a background in DNA damage/repair and/or DNA replication stress response. They should have a commitment to research in cancer biology, replication stress and R-loop biology and should hold a minimum upper second-class honours (2:1) degree in Biomedical Science, Biology, Biochemistry or any related discipline. A Master’s degree is desirable. 

For informal discussions and queries please contact Dr Panagiotis Kotsantis [email protected]  

To apply, please send your CV (max 2 pages) including the names of two referees and a cover letter (outlining your interest in this PhD and qualifications) to Dr Panagiotis Kotsantis [email protected]