DNA replication and repair in microcephalic dwarfism

Human height varies greatly between and within populations, and some individuals fall at the extreme ends of this wide spectrum. At the lower end of this distribution, individuals demonstrating extreme prenatal-onset reduction in body size and brain growth are classified as having microcephalic primordial dwarfism (MPD), which encompasses a group of rare single-gene disorders, usually inherited in an autosomal recessive manner. The human brain is particularly susceptible to perturbation during embryonic development, and the inability of neural progenitor cells to complete timely proliferation is thought to be an important contributor to the observed reduction in cerebral cortical size. Studying genes whose disruption leads to severe reduction in human growth can facilitate our understanding of the molecular pathways underlying cell proliferation and organism development. Mutations in many identified MPD genes result in the extended length of the cell cycle and impaired cell division by affecting essential cellular processes, such as DNA replication, DNA damage response (DDR) signalling, centriole biogenesis and mRNA splicing. The ability of cells to efficiently copy DNA and maintain the stability of their genome by promoting error-free repair of various types of DNA damage caused by endogenous and exogenous sources is particularly important for the timely cell cycle completion and cell survival. Therefore, it is not surprising that many MPD genes play a role in DNA replication, DDR and DNA repair. In this thesis, three DNA replication and DDR genes, mutated in MPD, are investigated. DNA2, encoding an ATP-dependent helicase/nuclease, was found to be mutated in four MPD patients. Experiments to confirm pathogenicity of the identified mutations indicated that they are likely to cause disease by affecting DNA2 transcript splicing and its enzymatic activities. My work described here also analyses the cellular role of TRAIP, an E3 ubiquitin ligase, which was linked to MPD by our laboratory (Harley et al., 2016). Cell experiments using TRAIP knockout cell lines, generated with CRISPR/Cas9 genome editing technology, demonstrated the requirement for TRAIP and its E3 ligase activity in DDR and repair of camptothecin (CPT)-induced DNA damage. Additionally, TRAIP was important for cell survival after mitomycin C (MMC)-induced DNA damage, but no epistasis with the Fanconi Anaemia (FA) interstrand crosslink (ICL) repair pathway was demonstrated, indicating an additive effect of TRAIP and FA-ICL pathways to repair these DNA lesions. Finally, generation of a mouse model of MPD caused by mutations in DONSON, a novel replication fork protection factor (Reynolds et al., 2017), is described in this thesis. DONSON MPD mice, harbouring the mouse equivalent of one of the human MPD missense mutations, showed embryonic lethality, with homozygous mutant embryos significantly smaller than their littermates and exhibiting limb abnormalities. Increased levels of spontaneous DNA damage were observed in mouse embryonic fibroblasts established from these embryos, mimicking the cellular phenotype of human DONSON deficiency. In summary, this thesis advances our knowledge of the cellular and developmental roles of MPD genes TRAIP, DNA2, and DONSON, that encode proteins maintaining genome stability

DNA Polymerase Epsilon Deficiency Causes IMAGe Syndrome with Variable Immunodeficiency.

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability

Identification of novel pathways linking epithelial-to-mesenchymal transition with resistance to HER2-targeted therapy

Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies in the treatment of HER2-positive breast cancer is a major clinical problem. To identify pathways linked to resistance, we generated HER2-positive breast cancer cell lines which are resistant to either lapatinib or AZD8931, two pan-HER family kinase inhibitors. Resistance was HER2 independent and was associated with epithelial-to-mesenchymal transition (EMT), resulting in increased proliferation and migration of the resistant cells. Using a global proteomics approach, we identified a novel set of EMT-associated proteins linked to HER2-independent resistance. We demonstrate that a subset of these EMT-associated genes is predictive of prognosis within the ERBB2 subtype of human breast cancers. Furthermore, targeting the EMT-associated kinases Src and Axl potently inhibited proliferation of the resistant cells, and inhibitors to these kinases may provide additional options for the treatment of HER2-independent resistance in tumors

CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.

The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically