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The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress

Raymond Buser1, Vanessa Kellner2, Andre Melnik1

  • 1Institute of Biochemistry, Department of Biology, ETH Zurich, Zürich, Switzerland.

Plos Genetics
|February 6, 2016
PubMed

Insights

Cullin 4-based E3 ubiquitin ligases (CRL4) are crucial for DNA replication and repair. The Rtt101(Mms22) complex counteracts Mrc1

Area of Science:

  • Molecular Biology
  • DNA Replication and Repair
  • Ubiquitin Ligases

Background:

  • Cullin 4-based E3 ubiquitin ligases (CRL4) are essential for faithful DNA replication and repair.
  • The specific mechanisms by which CRL4s function, particularly in response to DNA damage, are not fully understood.
  • The budding yeast Rtt101(Mms1, Mms22) complex is a CRL4 homolog involved in replication fork progression through damaged DNA.

Purpose of the Study:

  • To elucidate the molecular mechanisms of the Rtt101(Mms22) E3 ligase during DNA replication stress.
  • To identify interacting partners of Mms22 and understand its role in the replisome progression complex.
  • To investigate the functional relationship between Rtt101(Mms22) and the fork protection complex component, Mrc1.

Main Methods:

  • Interactome analysis of Mms22 to identify associated proteins.
  • Genetic screening for suppressors of genotoxic sensitivity in rtt101Δ cells.
  • Analysis of DNA replication and repair mechanisms in wild-type and mutant yeast strains under replication stress conditions.

Main Results:

  • The Rtt101(Mms22) ligase interacts with the replisome progression complex via Ctf4 during S-phase.
  • Genetic screens identified Mrc1, a component of the fork protection complex, as a key regulator interacting with Rtt101.
  • In contrast to single mutants, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants efficiently repair/restart stalled replication forks via a Rad52-dependent pathway.
  • The Rtt101(Mms22) E3 ligase appears to counteract Mrc1's function at stalled forks, potentially by modulating its interaction with the CMG complex, rather than inducing Mrc1 degradation.

Conclusions:

  • The Rtt101(Mms22) E3 ligase plays a specific role in managing replication fork progression during DNA damage.
  • Mrc1's function during replication stress is counteracted by the Rtt101(Mms22) ligase, revealing a novel regulatory axis.
  • This study sheds light on the coordinated action of CRL4s and fork protection complexes in maintaining genome stability.

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