RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans

Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans. Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint.

• Publication year

  2016

• Venue

  G3: Genes, Genomes, Genetics

• Publication date

  2016-08-18

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1534/g3.116.033043PMID 27543292PMCID 5068962
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• [Caenorhabditis elegans].

  2020cited by this paper
• Strategic role of the ubiquitin-dependent segregase p97 (VCP or Cdc48) in DNA replication

  2017cited by this paper
• Phosphopeptide binding by Sld3 links Dbf4‐dependent kinase to MCM replicative helicase activation

  2016influential reference
• MCM: one ring to rule them all.

  2016cited by this paper
• Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage*

  2016influential reference
• The mechanism of DNA replication termination in vertebrates

  2015cited by this paper
• Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins

  2015cited by this paper
• Termination of DNA replication forks: “Breaking up is hard to do”

  2015cited by this paper
• Polyubiquitylation drives replisome disassembly at the termination of DNA replication

  2014influential reference
• CDK phosphorylation of SLD-2 is required for replication initiation and germline development in C. elegans

  2014cited by this paper
• Phosphorylation of the BRCA1 C Terminus (BRCT) Repeat Inhibitor of hTERT (BRIT1) Protein Coordinates TopBP1 Protein Recruitment and Amplifies Ataxia Telangiectasia-mutated and Rad3-related (ATR) Signaling*

  2014influential reference
• Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication

  2014cited by this paper
• TopBP1: A BRCT-scaffold protein functioning in multiple cellular pathways.

  2014cited by this paper
• PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo

  2014influential reference
• CRL2LRR-1 E3-Ligase Regulates Proliferation and Progression through Meiosis in the Caenorhabditis elegans Germline

  2013influential reference
• Building and remodelling Cullin–RING E3 ubiquitin ligases

  2013cited by this paper
• BRCT domains: A little more than kin, and less than kind

  2012cited by this paper
• The dynamics of replication licensing in live Caenorhabditis elegans embryos

  2012cited by this paper
• Resource A Genome-Scale Resource for In Vivo Tag-Based Protein Function Exploration in C . elegans

  2012cited by this paper
• The structural basis for control of eukaryotic protein kinases.

  2012cited by this paper
• ATR signalling: more than meeting at the fork.

  2011cited by this paper
• MCM2-7 Form Double Hexamers at Licensed Origins in Xenopus Egg Extract

  2011cited by this paper
• CDC-48/p97 coordinates CDT-1 degradation with GINS chromatin dissociation to ensure faithful DNA replication.

  2011cited by this paper
• ATR autophosphorylation as a molecular switch for checkpoint activation.

  2011cited by this paper
• How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?

  2010influential reference
• CDK-dependent complex formation between replication proteins Dpb11, Sld2, Pol (epsilon}, and GINS in budding yeast.

  2010cited by this paper
• The CRL2LRR-1 ubiquitin ligase regulates cell cycle progression during C. elegans development

  2010cited by this paper
• Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling

  2010cited by this paper
• Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins.

  2010cited by this paper
• Regulation of cullin-RING E3 ubiquitin-ligases by neddylation and dimerization

  2009cited by this paper
• TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

  2009cited by this paper
• Using RNA Interference to Identify Specific Modifiers of a Temperature-Sensitive, Embryonic-Lethal Mutation in the Caenorhabditis elegans Ubiquitin-Like Nedd8 Protein Modification Pathway E1-Activating Gene rfl-1

  2009cited by this paper
• Targeting NEDD8-Activated Cullin-RING Ligases for the Treatment of Cancer

  2009cited by this paper
• A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication

  2009cited by this paper
• Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing.

  2009cited by this paper
• A Versatile Nanotrap for Biochemical and Functional Studies with Fluorescent Fusion Proteins*S

  2008cited by this paper
• HTP-3 links DSB formation with homolog pairing and crossing over during C. elegans meiosis.

  2008cited by this paper
• How ATR turns on: TopBP1 goes on ATRIP with ATR.

  2008influential reference
• TopBP1 activates ATR through ATRIP and a PIKK regulatory domain.

  2008influential reference
• Cell cycle progression requires the CDC-48UFD−1/NPL−4 complex for efficient DNA replication

  2008cited by this paper
• ATR: an essential regulator of genome integrity

  2008cited by this paper
• The Rad9-Hus1-Rad1 Checkpoint Clamp Regulates Interaction of TopBP1 with ATR*

  2007cited by this paper
• The DNA damage response: ten years after.

  2007cited by this paper
• Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast

  2007cited by this paper
• CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast

  2007cited by this paper
• The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1.

  2007cited by this paper
• Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells.

  2007cited by this paper
• TopBP1 activates the ATR-ATRIP complex.

  2006cited by this paper
• Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling

  2006cited by this paper
• Distinct modes of ATR activation after replication stress and DNA double‐strand breaks in Caenorhabditis elegans

  2005cited by this paper
• The pST44 polycistronic expression system for producing protein complexes in Escherichia coli.

  2005cited by this paper
• Systematic, RNA-Interference-Mediated Identification of mus-101 Modifier Genes in Caenorhabditis elegans

  2005cited by this paper
• Preventing re-replication of chromosomal DNA

  2005influential reference
• Identification and functional analysis of TopBP1 and its homologs.

  2005cited by this paper
• Chromosome sites play dual roles to establish homologous synapsis during meiosis in C. elegans.

  2005cited by this paper
• Claspin and the Activated Form of ATR-ATRIP Collaborate in the Activation of Chk1*

  2004influential reference
• CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing

  2003cited by this paper
• [C. elegans: of neurons and genes].

  2003cited by this paper
• Differential activation of the DNA replication checkpoint contributes to asynchrony of cell division in C. elegans embryos.

  2003cited by this paper
• The BRCT Domain Is a Phospho-Protein Binding Domain

  2003cited by this paper
• Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes

  2003cited by this paper
• Systematic functional analysis of the Caenorhabditis elegans genome using RNAi

  2003cited by this paper
• Identification of microcephalin, a protein implicated in determining the size of the human brain.

  2002cited by this paper
• Synapsis-dependent and -independent mechanisms stabilize homolog pairing during meiotic prophase in C. elegans.

  2002cited by this paper
• Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.

  2001cited by this paper
• ATR and ATRIP: Partners in Checkpoint Signaling

  2001cited by this paper
• A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli.

  2001cited by this paper
• Functional genomic analysis of C. elegans chromosome I by systematic RNA interference

  2000cited by this paper
• Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans

  2000cited by this paper
• DNA replication defects delay cell division and disrupt cell polarity in early Caenorhabditis elegans embryos.

  2000cited by this paper
• Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts.

  2000influential reference
• Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts.

  2000cited by this paper
• Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.

  2000cited by this paper
• Time-dependent responses to glp-1-mediated inductions in early C. elegans embryos.

  1996cited by this paper
• [Genetics of Caenorhabditis elegans].

  1989cited by this paper
• The genetics of Caenorhabditis elegans.

  1974cited by this paper

• DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in C. elegans

  2023cites this paper
• Replication stress promotes cell elimination by extrusion

  2021cites this paper
• Genetic analysis of Caenorhabditis elegans pry-1/Axin suppressors identifies genes involved in reproductive structure development, stress responses, and aging

  2021cites this paper
• The Fate of Two Unstoppable Trains After Arriving Destination: Replisome Disassembly During DNA Replication Termination

  2021cites this paper
• Cell cycle S-phase arrest drives cell extrusion

  2019cites this paper
• Terminating the replication helicase

  2017cites this paper
• Dynamic ubiquitin signaling in cell cycle regulation

  2017cites this paper
• CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination

  2017cites this paper
• Dynamic ubiquitin signaling in cell cycle regulation

  2017cites this paper
• Conducting the finale of DNA replication.

  2017cites this paper
• Termination of Eukaryotic Replication Forks.

  2017cites this paper
• CUL-2LRR-1 and UBXN-3/FAF1 drive replisome disassembly during DNA replication termination and mitosis

  2017cites this paper
• Dynamic ubiquitin signaling in cell cycle regulation

  2017cites this paper