View Program - 63rd Annual Drosophila Research Conference

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 assists by directly binding to BLM and helping to release the torsional stress on DNA as BLM helicase 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. To investigate the Blm-Top3α interaction in DNA repair, I performed a yeast 2-hybrid (Y2H) assay using the Drosophila proteins. I found interaction was specific to certain regions of Blm, with the strongest observed for a C-terminal region conserved among several Drosophila species, amino acids (aa) 1381-1487. To further decipher Blm roles, I created specific Blm deletions of regions from my Y2H data via CRISPR/Cas9 editing and tested various Blm roles in vivo. First I examined the role of Blm in promoting embryonic development. While deletion of aa 1-240 did not have an effect, deletion of aa 576-720 had substantial impact with most embryos failing to hatch. I then assessed the effects of Blm deletions on known Blm roles in meiotic chromosome segregation via a nondisjunction assay. Both aa 1-240 and aa 576-720 deletions were not significantly different from our wild-type control in contrast to my embryonic development data. This result suggests deletion of aa 576-720 is a separation-of-function mutation. Future studies will assess importance of these Blm regions in preventing mitotic crossovers and determining their importance to Blm function in DNA repair. I will also further explore Blm aa 576-720 and the relevance of predicted ATR/ATM (Drosophila Mei-41 and Tefu; regulators of DNA repair) phosphorylation sites within this region for proper Blm function in embryonic development, meiotic segregation, and DNA repair. By characterizing the functions of Blm in Drosophila, we will better understand BLM function within humans and the detrimental health effects of BLM mutations.

Functional Analysis of Bloom Syndrome Helicase in Development and DNA Repair