An analysis of the S. cerevisiae RMI1 gene

Thesis

The Saccharomyces cerevisiae Rmi1 protein is a component of the highly conserved Sgs1-Top3-Rmi1 complex, which is required for the maintenance of genome stability. The rmi1Δ deletion mutant has proven difficult to study because it exhibits very poor growth, and rapidly accumulates second site suppressor mutations. Furthermore, deletion of the putative HJ resolvase genes, MUS81-MUS81 or SLX1-SLX4 in rmi1Δ mutants causes synthetic lethality. In order to study phenotypes caused by loss of functional Rmi1, and to explore the genetic interactions between RMI1 and the MUS81, MUS81, SLX1 and SLX4 genes, a temperature sensitive mutant of RMI1 was isolated, named rmi1-1. Similar to rmi1Δ deletion mutants, rmi1-1 cells are highly sensitive to the DNA damaging agent, MMS and the replication inhibitor, HU. In addition, rmi1-1 mutants accumulate replication-associated branched DNA structures, and arrest in G₂/M after a transient exposure to MMS. These cells are proficient in DNA damage checkpoint activation. Deletion of SLX1, SLX4, MUS81 or MUS81 in the rmi1-1 strain causes synthetic lethality, which is associated with cell cycle defects. Following a transient exposure to MMS, rmi1-1 mutants accumulate homologous recombination intermediates. These intermediates are slowly resolved at the restrictive temperature, revealing a redundant resolution activity in the absence of functional Rmi1. This resolution depends upon Mus81-Mms4, but not on Slx1-Slx4 or Yen1. I propose that while the Sgs1-Top3-Rmi1 complex constitutes the main pathway for removal of homologous recombination intermediates following a perturbed S-phase, Mus81-Mms4 can act as a back up for resolution of these intermediates, which most likely represent double Holliday junctions. In this study, I also present screens for high copy suppressors of rmi1-1 phenotypes, and for novel Rmi1 interaction partners.

Authors: Ashton, TM

• Publication date: 2010
• DOI: 10.5287/ora-j5baqd7vp
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Rmi1
• Subjects: Oncology; DNA damage signalling; Genetics (medical sciences)