The replisome-coupled E3 ubiquitin ligase Rtt101Mms22 counteracts Mrc1 function to tolerate genotoxic stress

Raymond Buser; Vanessa Kellner; Andre Melnik; Caroline Wilson-Zbinden; René Schellhaas; Lisa Kastner; Wojciech Piwko; Martina Dees; Paola Picotti; Marija Maric; Karim Labib; Brian Luke; Matthias Peter

doi:10.1371/journal.pgen.1005843

The replisome-coupled E3 ubiquitin ligase Rtt101Mms22 counteracts Mrc1 function to tolerate genotoxic stress

Raymond Buser, Vanessa Kellner, Andre Melnik, Caroline Wilson-Zbinden, René Schellhaas, Lisa Kastner, Wojciech Piwko, Martina Dees, Paola Picotti, Marija Maric, Karim Labib, Brian Luke (Lead / Corresponding author), Matthias Peter (Lead / Corresponding author)

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Abstract

Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1's replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.

Original language	English
Article number	e1005843
Number of pages	25
Journal	PLoS Genetics
Volume	12
Issue number	2
DOIs	https://doi.org/10.1371/journal.pgen.1005843
Publication status	Published - 5 Feb 2016

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10.1371/journal.pgen.1005843Licence: CC BY

journal.pgen.1005843Final published version, 9.82 MBLicence: CC BY

Functional Dissection of the Eukaryotic Replisome (Senior Investigator Award)
Labib, K. (Investigator)
1/04/14 → 29/02/20
Project: Research

Labib, Karim
- Genome Integrity - Professor of Genome Integrity
Person: Academic

Cite this

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title = "The replisome-coupled E3 ubiquitin ligase Rtt101Mms22 counteracts Mrc1 function to tolerate genotoxic stress",

abstract = "Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1's replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.",

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AU - Buser, Raymond

AU - Kellner, Vanessa

AU - Melnik, Andre

AU - Wilson-Zbinden, Caroline

AU - Schellhaas, René

AU - Kastner, Lisa

AU - Piwko, Wojciech

AU - Dees, Martina

AU - Picotti, Paola

AU - Maric, Marija

AU - Labib, Karim

AU - Luke, Brian

AU - Peter, Matthias

PY - 2016/2/5

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N2 - Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1's replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.

AB - Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1's replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.

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The replisome-coupled E3 ubiquitin ligase Rtt101Mms22 counteracts Mrc1 function to tolerate genotoxic stress

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Functional Dissection of the Eukaryotic Replisome (Senior Investigator Award)

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Labib, Karim

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