Germline mutations affecting the RECQL4 DNA helicase cause Type II Rothmund-Thomson syndrome (RTS), a human disease characterised by defects in skeletal development and predisposition to specific types of cancer, including osteosarcoma (OS). RECQL4 has been implicated in multiple cellular functions that mediate accurate DNA replication and repair. How germline RECQL4 mutations associated with Type II RTS affect these functions to cause disease remains unclear, in part due to the paucity of appropriate cellular models.

In this work, CRISPR/Cas9 gene editing was used to generate cell lines containing a prevalent RTS patient RECQL4 mutation, the “Mut-2” c.2269C>T. The resulting Mut-2 clones exhibited greatly reduced RECQL4 protein levels, similar to decreases observed in RTS patient cells. Unexpectedly, the major effect of this predicted nonsense mutation was the upregulation of the use of an alternative splice site in exon 14 which skipped the premature stop codon and resulted in the deletion of 66 amino acids in the RECQL4 ATPase domain.

Despite the lower overall RECQL4 expression, single cell clones bearing the Mut-2 mutation showed mostly normal cell cycle distribution with a slight increase in population doubling times. When challenged with various DNA damaging agents, these Mut-2 clones exhibited increased sensitivities to DNA alkylators and topoisomerase inhibitors, and mild sensitivities to DNA crosslinkers and PARP inhibitors, a sensitivity profile suggestive of defects in DNA double-strand break (DSB) repair.

When further assayed using flow cytometric GFP reporters, the Mut-2 clones showed decreased DNA DSB repair capacities in the homologous recombination (HR) and microhomology mediated end joining (MMEJ) pathways, providing evidence that RECQL4 disruption impacted replication-specific DNA DSB repair in particular. Additional RECQL4 reconstitution studies confirmed that the decreased HR repair was a result of structural changes to RECQL4 due to the Mut-2 mutation.

Finally, the formation of RAD51 foci—a commonly used marker of HR function—in the Mut-2 clones post-DNA DSB induction was investigated. Surprisingly, upon DNA DSB challenge, all Mut-2 clones were as proficient at forming RAD51 foci as parental HEK293. This suggested that the RECQL4 Mut-2 mutation disrupted its function further downstream in the HR pathway than had been previously reported.

The work presented in this dissertation is a novel approach to studying the effects of clinical RTS RECQL4 mutations. These studies have illuminated mechanisms of RECQL4 disruption in Type II RTS as well as the roles of the RECQL4 helicase in cellular DNA damage repair. Because about 30% of Type II RTS patients are diagnosed with osteosarcoma, a common and deadly primary malignancy of the bone, the results presented here could shed new light on potential mechanisms underlying osteosarcoma tumour development and ultimately suggest new avenues and strategies for targeted clinical intervention.