A new method, “reverse yeast two-hybrid array” (RYTHA), identifies mutants that dissociate the physical interaction between Elg1 and SIx5

Ifat Lev, Keren Shemesh, Marina Volpe, Soumitra Sau, Nelly Levinton, Maya Molco, Shivani Singh, Batia Liefshitz, Shay Ben Aroya, Martin Kupiec

Research output: Contribution to journalArticlepeer-review


The vast majority of processes within the cell are carried out by proteins working in conjunction. The Yeast Two-Hybrid (Y2H) methodology allows the detection of physical interactions between any two interacting proteins. Here, we describe a novel systematic genetic methodology, “Reverse Yeast Two-Hybrid Array” (RYTHA), that allows the identification of proteins required for modulating the physical interaction between two given proteins. Our assay starts with a yeast strain in which the physical interaction of interest can be detected by growth on media lacking histidine, in the context of the Y2H methodology. By combining the synthetic genetic array technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify trans-acting mutations that disrupt the physical interaction of interest. We apply this novel method in a screen for mutants that disrupt the interaction between the N-terminus of Elg1 and the Slx5 protein. Elg1 is part of an alternative replication factor C-like complex that unloads PCNA during DNA replication and repair. Slx5 forms, together with Slx8, a SUMO-targeted ubiquitin ligase (STUbL) believed to send proteins to degradation. Our results show that the interaction requires both the STUbL activity and the PCNA unloading by Elg1, and identify topoisomerase I DNA-protein cross-links as a major factor in separating the two activities. Thus, we demonstrate that RYTHA can be applied to gain insights about particular pathways in yeast, by uncovering the connection between the proteasomal ubiquitin-dependent degradation pathway, DNA replication, and repair machinery, which can be separated by the topoisomerasemediated cross-links to DNA.

Original languageEnglish
Pages (from-to)1683-1697
Number of pages15
Issue number3
StatePublished - Jul 2017

Bibliographical note

Funding Information:
We thank the members of the S.B.A. and M.K laboratories for ideas and support. Research in M.K.’s laboratory was supported by grants from the Israel Science Foundation (ISF) and the Israel Cancer Research Fund (ICRF). Research in S.B.A.’s laboratory was supported in part by the Israel Science Foundation (grant number 49/12), Israel Cancer Research Fund (project grant 2015–16), and the Israel Cancer AssociationICA (grant number 20161150).

Publisher Copyright:
© 2017 by the Genetics Society of America.


  • Clamp unloader
  • PCNA
  • SGA
  • SUMO-targeted ubiquitin ligase (STUbL)


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