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Novel ubiquitin ligases and DUBs in DNA replication and damage

Principal Supervisor: Dr Aga Gambus - Institute of Cancer and Genomic Sciences

Co-supervisor: Dr Mark Wigglesworth

PhD project title: Novel ubiquitin ligases and DUBs in DNA replication and damage

University of Registration: Birmingham

Project outline:

Faultless DNA replication is essential for genomic stability. We have shown that ubiquitylation drives disassembly of replication machinery at the end of DNA replication opening a new area of research. This project aims to characterise ubiquitin ligases and deubiquitylting enzymes identified to interact with replication forks or with terminating replication machinery (Dungrawala at al 2015 and our unpublished data). All of these enzymes are frequently de-regulated in cancer. We will determine the function of these enzymes during DNA replication and damage response (DDR) and assess as potential therapy targets. To accomplish this we will utilise a combination of biochemical approaches using cell free Xenopus laevis egg extract and cell biology techniques in human immortalised cell lines.

Objective 1: Characterise when studied enzymes bind chromatin and their potential substrates

We will raise antibodies against studied enzymes (ahead of project start) and study their interaction with replisome during DNA replication and DDR. We will also purify inactive mutants of these enzymes – with mutated active sites whilst still able to recognise substrates, and use them as a bait to identify interacting partners by mass spectrometry. These will represent potential substrates, which we will analyse and confirm.

Objectove 2: Understand the function and mechanism of action of studied enzymes during DNA replication and damage

We will then determine the mechanism of action of these enzymes during DDR. We will assess whether the substrates can bind/disassemble normally during repair in absence of modifying enzyme and if the repair can be performed. We will also use known inhibitors of DDR to study their interplay with studied enzymes. The X.l. extract provides a simple cell free model system to study DNA replication and DDR, is highly synchronous and easy to manipulate biochemically – providing a key advantage for the delivery of the first two objectives.

Objective 3: Determine phenotype of down-regulation of these enzymes in human cells and its link with DNA replication/damage

We will investigate the phenotype of down-regulation of studied enzymes on a cellular level using normal immortalised retina pigment epithelium (RPE) and cancerous (but chromosomally stable) osteosarcoma call line (U2OS). While carrying out objectives 1 and 2 we will knock out our enzymes of interest (or tag essential genes with AID-degron tag) using CRISPR. We will use the created cell lines to determine the effect of the enzymes depletion on cellular proliferation and on DDR.

Objective 4: Investigate differential requirements for the studied enzymes in normal and cancerous cell lines

Finally we will examine whether the studied enzymes could serve as potential therapy targets. We will compare the effect of siRNA downregulation of studied enzymes amongst a panel of selected cancer cell lines chosen based on their reported de-regulation in c-bioportal and access further DDR models available within AstraZeneca.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

The student will work both with human cell lines as well as Xenopus laevis egg extract system and henceforth learn techniques specific for both. It will provide opportunity to carry on proteomic analysis of chromatin bound complexes in egg extract system but also DNA analysis and microscopy in human cell lines.

Contact: Dr Aga Gambus, University of Birmingham