Formation of a Chimeric Gene in Exploring the Functional Conservation of Mms21

Abstract:

The structural maintenance of chromosomes 5/6 complex is the least understood complex within the Smc protein family. An interesting subunit of this complex within Saccharomyces cerevisiae is Mms21, which is a small ubiquitin-like modifier E3 ligase thought to be responsible for regulating the sumoylation of other proteins involved in DNA repair mechanisms and checkpoint signaling. The contribution of the ligase activity of Mms21 to genomic stability is exemplified by notable phenotypes of ligase-deficient mutants in comparison to wild-type strains, including hypersensitivity to DNA-damaging agents, as well as the evolutionary conservation of the Mms21-ligase domain between species. While many homologues of Mms21 have already been identified, a less established homologue is the protein encoded by the Dictyostelium discoideum gene DDB_G0289341. Within this project, I aim to confirm the protein encoded by DDB_G0289341 as an orthologue of Mms21, and to explore the interchangeability of the ligase domain of Mms21 between species. Such knowledge would enhance the significance of the role of Mms21 in genomic stability, and may lead to an increased urgency to elucidate the contributions of Mms21 and the Smc5/6 complex to human pathologies. My hypothesis is that the incorporation of the DNA sequence encoding for the C-terminal region of the protein encoded by DDB_G0289341 into the MMS21 ligase-domain sequence, forming a chimeric gene, will result in Mms21 ligase activity within the chimeric strain, suggesting functional conservation between species. Within this project, I have successfully formed the chimeric gene through HiFi assembly and yeast transformation. The Mms21-ligase activity of the chimeric strain will be determined through spotting assays, in which the growth of the chimeric strain following exposure to DNA-damaging agents (ultraviolet radiation and methyl methanesulfonate) will be compared to both wild-type and mms21-CH (ligase deficient) mutant strains.