Functional Analysis of Bloom Syndrome Helicase in Development and DNA Repair

Bloom Syndrome is a rare autosomal recessive disorder in humans caused by mutation of the BLM gene that leads to increased genome instability and cancer. The BLM gene codes for a helicase (BLM) that works together with Topoisomerase 3-alpha (Top3α) in homology-directed repair of DNA. Top3α directly binds to BLM and helps release the torsional stress on DNA as BLM unwinds recombination intermediates. These proteins preserve genome stability and have been shown in many organisms to operate together in the prevention of detrimental mitotic (non-meiotic) crossovers via two main DNA repair pathways, synthesis-dependent strand annealing and double Holliday junction dissolution. In Drosophila, BLM (known as Blm) also has roles in proper meiotic chromosome segregation and rapid cell cycle progression of the developing embryo. Each of these BLM functions are not well characterized and limit development of treatments for BLM-related disorders. To investigate the Blm-Top3α interaction in DNA repair, I performed a yeast 2-hybrid (Y2H) assay using the Drosophila genes. I found the interaction was specific to certain regions of Blm, with the strongest interaction observed at a C-terminal region conserved among several Drosophila species, amino acids (aa) 1381-1487. Based on these data, I created specific deletions of the Blm gene via CRISPR/Cas9 editing to characterize the various roles of Blm in vivo. First, I assessed the effects of Blm deletions on known Blm roles in meiotic chromosome segregation via a nondisjunction assay. Both aa 576-720 and N1 produced measurable defects compared to the wild type suggesting functional value of aa 576-720. Significance of this Blm region in preventing mitotic crossovers and DNA repair was evaluated by a crossover assay showcasing a lack of significant effect by aa 576-720 relative to the true null allele, N1, but still increased relative to wild type. These studies showcase the importance of aa 576-720 and other Blm regions in the roles of meiotic segregation and DNA repair. Blm aa 576-720 will be further assessed by examining the relevance of predicted ATR/ATM phosphorylation sites within the region required for proper Blm function. Additional Blm roles in embryonic development will also be explored via an embryo hatching assay. By characterizing the functions of Blm in Drosophila, we will better understand and improve BLM function within humans and the detrimental health effects associated with BLM mutations.Bachelor of Scienc

Functional Analysis of Bloom Syndrome Helicase in Development and DNA Repair

Bloom Syndrome is a rare autosomal recessive disorder in humans caused by mutation of the BLM gene that leads to increased genome instability and cancer. The BLM gene codes for a helicase (BLM) that works together with Topoisomerase 3-alpha (Top3α) in homology-directed repair of DNA. Top3α directly binds to BLM and helps release the torsional stress on DNA as BLM unwinds recombination intermediates. These proteins preserve genome stability and have been shown in many organisms to operate together in the prevention of detrimental mitotic (non-meiotic) crossovers via two main DNA repair pathways, synthesis-dependent strand annealing and double Holliday junction dissolution. In Drosophila, BLM (known as Blm) also has roles in proper meiotic chromosome segregation and rapid cell cycle progression of the developing embryo. Each of these BLM functions are not well characterized and limit development of treatments for BLM-related disorders. To investigate the Blm-Top3α interaction in DNA repair, I performed a yeast 2-hybrid (Y2H) assay using the Drosophila genes . I found the interaction was specific to certain regions of Blm, with the strongest interaction observed at a C-terminal region conserved among several Drosophila species, amino acids (aa) 1381-1487. Based on this data, I created specific deletions of the Blm gene via CRISPR/Cas9 editing to characterize the various roles of Blm in vivo. First, I assessed the effects of Blm deletions on known Blm roles in meiotic chromosome segregation via a nondisjunction assay. Both aa 576-720 and N1 produced measurable defects compared to the wild type suggesting functional value of aa 576-720. Significance of this Blm region in preventing mitotic crossovers and DNA repair was evaluated by a crossover assay showcasing a lack of significant effect by aa 576-720 relative to the true null allele, N1, but still increased relative to wild type. These studies showcase the importance of aa 576-720 and other Blm regions in the roles of meiotic segregation and DNA repair. Blm aa 576-720 will be further assessed by examining the relevance of predicted ATR/ATM phosphorylation sites within the region required for proper Blm function. Additional Blm roles in embryonic development will also be explored via an embryo hatching assay. By characterizing the functions of Blm in Drosophila, we will better understand and improve BLM function within humans and the detrimental health effects associated with BLM mutations

Mis-spliced transcripts generate de novo proteins in TDP-43–related ALS/FTD

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43–depleted human iPSC–derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43–depleted human iPSC–derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43–depleted human iPSC–derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF