Drosophila Mage, a component of Smc5/6 DNA response complex, confers resistance to caffeine and genotoxic stress and plays a role in the cell cycle and cell survival

• Author / Creator

  Li, Xiao

• Over 50 Melanoma-associated antigen (MAGE) genes have been identified in the human genome. They share a conserved 200 amino acid MAGE-homology domain (MHD). In yeast, the only MAGE homolog (Nse3) is a component of the Smc5/6 DNA damage response complex. In humans, MAGE proteins influence cell cycle and cell survival via interaction with Rb-E2F and p53 pathways and some MAGE proteins interact with the human Smc5/6 complex as well. Only one MAGE gene (MAGE) exists in the genome of the fruit fly Drosophila melanogaster.
  We hypothesize that the functions of the MAGE proteins are conserved during evolution. Drosophila Mage is part of Smc5/6 complex and plays a role in the DNA damage response. It may also regulate the cell cycle and cell survival by interacting with Drosophila p53 (Dmp53).
  We found that Mage bound to Smc5/6 components (i.e. Nse1 and Nse4) in co-immuno-precipitation and in vitro pull-down experiments. We generated Drosophila mutants of MAGE, Smc5, Smc6, and Nse1 and found that all mutants were viable, but hypersensitive to caffeine and genotoxic agents. We also studied the effects of Mage over-expression in a Drosophila cell line and found that over-expression of Mage slows cell proliferation by arresting cells in S and M phases. Further, over-expression of Mage also confers a growth advantage to cells exposed to genotoxic stress. We also found an interdependency of protein stability between Mage and its interaction partners including Smc5, Smc6, Nse4, Nse1 and p53. Finally, unlike a human MAGE homolog, Necdin, which interacts with p53 directly, we found that Mage does not interact with Dmp53, however, Nse4 and Dmp53 associate.
  Like other Smc5/6 gene mutants, MAGE mutants are also caffeine-sensitive and Mage physically interacts with the Drosophila homologs of the Nse proteins suggesting that the structure of the Smc5/6 complex is conserved in Drosophila. Although Smc5/6 proteins are required for viability in S. cerevisiae, they are not essential under normal circumstances in Drosophila. However, flies carrying mutations in Smc5, Smc6, Nse1 and MAGE are hypersensitive to genotoxic agents, consistent with a role for the Smc5/6 complex in genome stability. Like Necdin, over-expression of Mage inhibits cell proliferation and promotes cell survival. This result could be explained by the direct interaction with the Smc5/6 complex and the indirect interaction with Dmp53.
  This study reveals a conserved role of Mage as a part of Smc5/6 DNA response complex in maintaining genome stability. It also hints at a functional link between p53 and the Smc5/6 complex. Together, these data will help to uncover how this expanded protein family plays such versatile roles in cancer and development in humans.

• Subjects / Keywords

  • Nse1
  • Genotoxic stress
  • DNA damage
  • ATR
  • Nse2
  • Nse4
  • Smc5
  • Cell cycle
  • Rad51
  • Cell survival
  • Drug resistance
  • Caffeine
  • ATM
  • Smc6
  • NBS1
  • MAGE
  • Nse3
  • Drosophila

• Graduation date

  Fall 2014

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3Q68G

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.