Using S. cerevisiae to screen cancer mutations in the DNA damage response pathway

Abstract

When DNA is damaged, a DNA damage response (DDR) pathway is initiated to prevent mutations from being passed on to daughter cells. The MRN complex in humans (MRX in yeast) functions to start the DDR pathway by detecting DNA damage and is comprised of Mre11, Rad50, and Nbs1 in humans. Although mutations in members of the MRN complex pre‐dispose individuals to cancer, the functional effects of Mre11 cancer mutations have not been well characterized. The goal of this project was to investigate the effects of Mre11 cancer mutations on the DDR pathway using yeast‐based assays because the complex is highly conserved in eukaryotes. Using a database of human tumor mutations, we identified 233 cancer mutations in human Mre11, 83 of which occurred at residues that are conserved between yeast and human. A subset of these mutations were screened for functional effects in yeast by creating the cancer mutations at the conserved residues in yeast Mre11. Yeast expressing wild‐type or mutant Mre11 were then assessed for DNA damage sensitivity using hydroxyurea (HU) and methyl methanesulfonate (MMS). Results indicate that N‐terminal point mutations in yeast Mre11 do not confer sensitivity to DNA damage in yeast cells suggesting that these regions may have conserved sequence without conserved function. However, one mutation near the capping domain of Mre11 does show the disfunction of the yeast Mre11 protein similar to the deletion strain. Using yeast‐based assays, cancer mutations can rapidly be screened for functional effects on DDR pathways. Support or Funding Information Longwood University Faculty Research and Development Grant