UBC Theses and Dissertations
Unravelling RecQ helicase function in genome stability using Strand-seq Hamadeh, Zeid
Helicases are a highly conserved family of motor proteins responsible for interacting with and unwinding canonical and non-canonical DNA and RNA structures. The RecQ class of helicases, known to suppress illegitimate recombination, are implicated in aging and cancer with four of the five human RecQ helicases directly linked to genome instability syndromes characterized in some cases by strong cancer predisposition or premature aging. While no human disease has been associated with the RECQL5 helicase, loss of this gene in cells is known to result in elevated double strand breaks (DSBs) and sister chromatid exchange events (SCEs), a phenotype of genome instability similar to what is observed in RecQ helicase-linked diseases of strong cancer predisposition. Until recently, studying SCEs has been limited to cytogenetic assays that map at megabase resolution. I used single cell template strand sequencing (Strand-seq) to map SCEs as changes in template strand orientation before and after loss of RECQL5 at kilobase resolution. I generated over 20 single and double knockout models for RECQL5 as well as BLM, WRN and RECQL1 helicases using CRISPR-Cas9 in the human haploid cell line, KBM7, and mapped SCEs to the genome using custom bioinformatic approaches to improve resolution and accuracy of SCE detection. I performed enrichment analysis to show SCEs are frequently occurring near actively transcribed genes with guanine quadruplexes (G4s) and common fragile sites further supporting the role of these helicase genes in suppressing inappropriate recombination at specific genomic elements. I also developed novel bioinformatic approaches to generate genotype-specific call sets for copy number alterations (CNAs), inversions, and translocations. Uncovering the role of DNA helicases in DNA repair and replication pathways is critical for understanding their significance in cancer and aging. Stand-seq offers a unique method to study helicases by mapping the location of SCEs arising in their absence.
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