RECQL4; Linking DNA Replication and Bone Tumourigenesis Tammy Wiltshire A thesis submitted in partial fulfilment of the requirements of the University of Lincoln for the degree of Doctor of Philosophy School of Life Sciences October 2019 Abstract As part of cell division, the initiation of DNA replication is an important regulatory mechanism that maintains genomic stability over generations. RECQL4 belongs to the RecQ DNA helicase family holding an important role in the initiation of DNA replication. RECQL4 mutations can lead to disorders including type II Rothmund- Thomson (RTS) syndrome. These patients demonstrate predisposition to osteosarcoma (OS) development. OS is a primary bone tumour showing extensive chromosomal instability. We hypothesise mesenchymal stem cells (MSC) differentiating to osteoblasts are particularly sensitive to RECQL4 mutations, which may lead to impaired differentiation and OS. Our aim is to establish a direct link between the impairment of replication initiation, consequent chromosomal instabilities and deregulation in osteoblast differentiation, and OS development. A model that phenocopies the effect of RECQL4 mutations was established in ASC52telo cells to apply acute and chronic pressure on replication initiation using PHA-767491, which inhibits DDK that acts upstream of RECQL4 in replication initiation (PHA cells). We monitored cell viability and chromosomal instability characteristics in these cells, and RTS cell lines. To establish if PHA cells sustained differentiation capability, the cells were cultured using supplemented media for osteoblast and adipocyte differentiation, and were analysed by histochemical staining and immunofluorescence. Presence of DNA damage was quantified using γH2AX, and activation of the DNA damage response was assayed by western blotting. The cells were cultured in ultra-low attachment plates to test for anchorage- independent growth, and further analysed by cell count, MTS and luminescence assays. To identify protein-protein interactions of RECQL4, GFP-tagged RECQL4 HeLa and U2OS cells were treated with SAHA, or hydroxyurea, pulled down with GFP-nanotrap, and analysed by mass spectrometry and western blot. We confirmed reduced proliferation rate while maintaining viability in PHA cells. Assaying mitochondrial membrane potential revealed no significant effect on mitochondrial function. Successful differentiation of PHA treated MSCs into osteoblasts and adipocytes was confirmed. Expression of osteoblast differentiation markers: calcium, and RUNX2 was influenced by PHA. An increase was also observed in chronic PHA cells under normal medium, indicating malignant transformation. Sustained DNA damage was shown in chronic PHA-767491 Page | i treated ASC52telo cells, with a higher degree of CHK1 phosphorylation, anchorage-independent growth and reduced contact inhibition. We found that the RECQL4 mutated cell line AG05013tert was more sensitive to inhibition of replication initiation. Increase of DNA damage markers was observed in AG05013tert cells, but not in AG18375 and AG03587. Presence of MCM10 and PP2A in RECQL4 complexes was confirmed, and novel interactions with HDX and EN-2 were found. Overall, we demonstrated that chronic interference with DNA replication initiation leads to sustained DNA damage with characteristics of genomic instability, activated DNA damage response that may become impaired over time, and may induce transformation. To further these studies, RECQL4 knockdown using lentiviral transduction in osteoblasts would verify the cellular changes we propose, which lead to chromosomal instabilities and OS development. Novel protein interactions with RECQL4 could highlight new pathways with a direct and/or indirect role in tumourgenesis. Page | ii Acknowledgements I would like to acknowledge and thank my supervisor Dr Csanád Z. Bachrati for all the support and encouragement within the project. I would like to acknowledge Timea Palmai-Pallag for producing the data required within the study, thank you so much to you both for your time you gave. I would also like to thank my family for all off their support. Page | iii Contents 1 Introduction ................................................................................................................ 1 1.1 Cell division ........................................................................................................... 1 1.1.1 Cell cycle ....................................................................................................... 1 1.1.1.1 Cyclin-dependent kinases ...................................................................... 2 1.1.2 Replication initiation ....................................................................................... 3 1.1.2.1 Replication initiation; RECQL4 ............................................................... 3 1.1.2.2 Impaired DNA replication initiation can lead to oncogenesis .................. 7 1.2 RECQL4 ................................................................................................................ 8 1.2.1 Biochemical activity of RECQL4 .................................................................. 10 1.2.2 RECQL4 localisation .................................................................................... 10 1.2.3 Mutations in the RECQL4 gene ................................................................... 11 1.2.3.1 Baller-Gerold (BGS) syndrome ............................................................. 12 1.2.3.2 RAPADILINO syndrome ....................................................................... 12 1.2.3.3 Rothmund Thomson Syndrome (RTS) ................................................. 12 1.2.3.3.1 Symptoms ......................................................................................... 13 1.2.3.3.2 Diagnosis ........................................................................................... 14 1.2.3.3.3 Subtypes ........................................................................................... 15 1.2.4 RTS and cancer predisposition .................................................................... 16 1.3 Mesenchymal stem cells ..................................................................................... 19 1.3.1 Bone formation ............................................................................................ 21 1.4 Primary bone malignancies ................................................................................. 25 1.4.1 Ewing sarcoma ............................................................................................ 25 1.4.2 Osteosarcoma ............................................................................................. 25 1.4.2.1 Cell cycle dysregulation and OS ........................................................... 27 1.4.2.2 OS and known gene involvements ....................................................... 28 1.4.2.2.1 P53 .................................................................................................... 28 1.4.2.2.2 RECQL4 and tumour development ................................................... 29 1.4.2.2.2.1 RECQL4 and OS ........................................................................ 29 1.4.2.2.3 RUNX2 and cancer ........................................................................... 30 1.4.2.3 OS and bone dysregulation .................................................................. 31 1.5 The DNA damage response and genome instability ........................................... 32 1.5.1 DNA damage ............................................................................................... 32 1.5.2 Hallmark features of genomic instability ...................................................... 33 1.5.3 Single stranded DNA ................................................................................... 34 1.5.4 Double stranded DNA break ........................................................................ 35 1.5.5 DNA repair pathways ................................................................................... 35 Page | iv 1.5.5.1 DNA damage response pathways ........................................................ 35 1.5.5.2 Homologous recombination .................................................................. 36 1.5.5.3 Non-homologous end-joining ................................................................ 38 1.5.5.4 Telomeres are disguised double-stranded breaks ............................... 40 1.5.5.5 Base excision repair ............................................................................. 44 1.5.5.6 Nucleotide excision repair .................................................................... 45 1.5.6 Involvement of RECQL4 in processes of DNA repair .................................. 47 1.6 Aims of project .................................................................................................... 50 2 Materials and Methods ............................................................................................ 51 2.1 Stock solutions .................................................................................................... 51 2.1.1 Stock solutions and chemical reagents ....................................................... 51 2.2 Cell Culture ......................................................................................................... 58 2.2.1 Cell maintenance ......................................................................................... 58 2.2.2 Freezing ......................................................................................................