Investigating the roles of DNA Polymerase Epsilon in genome stability

Primary Supervisor: Roberto Bellelli, Queen Mary University of London

Secondary supervisor: David Michod, UCL

Tertiary Supervisor: Peter Cherepanov, Francis Crick Institute

Project

Genomic instability is a major hallmark of cancer and understanding its nature has provided avenues for patient-tailored therapies. Recent studies uncovered DNA Polymerase Epsilon (hereafter Pol Epsilon), as a novel potential therapeutic target in cancer (1). In addition, genome wide CRISPR screenings have discovered that deletion of the POLE3-POLE4 subunits of Pol Epsilon leads to increased sensitivity to ATR and PARP inhibitors (2, 3). Aim of this project is to unveil the mechanisms behind these observations and exploit Pol Epsilon and POLE3-POLE4 as markers of sensitivity to PARP/ATR inhibitors and novel therapeutic targets. The project will combine approaches of biochemistry and structural biology, cell biology and bioinformatics, available within CRUK City of London Centre, to accomplish three aims:

Aim 1: Reconstitution of the dynamics of DNA replication and nucleosome assembly by Pol Epsilon
We previously discovered that POLE3-POLE4 bind histones H3-H4 and double stranded DNA (4); These activities might promote histone recycling at the replication fork and Pol Epsilon processivity, thus preventing gap accumulation at the replication fork. Loss of this function might then result in synthetic lethality with inhibition of PARP/ATR and the selective vulnerability of specific cancers (1). To address these hypotheses, we will study the dynamics of DNA synthesis and nucleosome assembly by Pol Epsilon in the presence or absence of POLE3-POLE4. In addition to this, we will generate a Cryo-EM structure of Pol Epsilon.

Aim 2: Identification of the roles of POLE3-POLE4 in DNA replication and the mechanism of synthetic lethality with ATR/PARP inhibitors
We recently showed an increased sensitivity of POLE4 KO cells to ATR and PARP inhibitors. To understand the mechanism behind this observation, we will analyse the dynamics of DNA replication in POLE3-POLE4 KO cells in the presence of ATR and PARP inhibitors by DNA fiber assay (5). In addition, we will monitor cell proliferation and apoptosis by Incucyte cell imaging and markers of replication stress and DNA damage (RPA, γH2AX, 53BP1) by confocal microscopy. Finally we will combine BrdU immunoprecipitation and Cut&Tag to analyse histone post-translational modifications at the replication fork in POLE3-POLE4 KO cells.

Aim 3: Analysis of Pol Epsilon expression in cancer
We will then combine bioinformatics and molecular biology to analyse expression levels of Pol Epsilon in cancer. In collaboration with the bioinformatic facilities at UCL, we will interrogate cancer databases for mRNA and protein expression levels of Pol Epsilon in cancer and correlate it with survival, tumour features and mutational signatures. Furthermore, we will assess the expression levels of POLE1, POLE3 and POLE4 in panels of ovarian and breast cancer cell lines presenting diverse sensitivity to PARP and ATR inhibitors. Importantly we have already generated the tools (e.g. specific antibodies) required to test the expression of Pol Epsilon subunits in cell lines and cancer tissues by IHC. Furthermore, we can access one of the major biobanks of cancer tissues in UK at BCI.

Candidate background

This project would suit candidates with a background in biochemistry and cell biology and an interest in genome stability.

Potential Research Placements

1. David Michod, UCL Institute of Child Health
2. Peter Cherepanov, Francis Crick Institute
3. Nnenne Kanu, UCL Cancer Institute

References

1. Sviderskiy VO, Blumenberg L, Gorodetsky E, Karakousi TR, Hirsh N, Alvarez SW, Terzi EM, Kaparos E, Whiten GC, Ssebyala S, Tonzi P, Mir H, Neel BG, Huang TT, Adams S, Ruggles KV, Possemato R. Hyperactive CDK2 Activity in Basal-like Breast Cancer Imposes a Genome Integrity Liability that Can Be Exploited by Targeting DNA Polymerase ε. Mol Cell 80, 682-698.
2. Hustedt N, Álvarez-Quilón A, McEwan A, Yuan JY, Cho T, Koob L, Hart T, Durocher D. A consensus set of genetic vulnerabilities to ATR inhibition. Open Biol. 9, 190156.
3. Su D, Feng X, Colic M, Wang Y, Zhang C, Wang C, Tang M, Hart T, Chen J. CRISPR/CAS9-based DNA damage response screens reveal gene-drug interactions. DNA Repair (Amst). 87, 102803
4. Bellelli R, Belan O, Pye VE, Clement C, Maslen SL, Skehel JM, Cherepanov P, Almouzni G, Boulton SJ. POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication. Mol. Cell 72,112-126.
5. Bellelli R*, Borel V*, Logan C, Svendsen J, Cox DE, Nye E, Metcalfe K, O’Connell SM, Stamp G, Flynn HR, Snijders AP, Lassailly F, Jackson A, Boulton SJ. Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis. Mol. Cell 70, 707-721. *equal contribution