title: Whole genome RNAi screens reveal a critical role of REV3 in coping with replication stress.
abstract: REV3, the catalytic subunit of translesion polymerase zeta (polζ), is commonly associated with DNA damage bypass and repair. Despite sharing accessory subunits with replicative polymerase δ, very little is known about the role of polζ in DNA replication. We previously demonstrated that inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells. To reveal determinants of this sensitivity and obtain insights into the cellular function of REV3, we performed whole human genome RNAi library screens aimed at identification of synthetic lethal interactions with REV3 in A549 lung cancer cells. The top confirmed hit was RRM1, the large subunit of ribonucleotide reductase (RNR), a critical enzyme of de novo nucleotide synthesis. Treatment with the RNR-inhibitor hydroxyurea (HU) synergistically increased the fraction of REV3-deficient cells containing single stranded DNA (ssDNA) as indicated by an increase in replication protein A (RPA). However, this increase was not accompanied by accumulation of the DNA damage marker γH2AX suggesting a role of REV3 in counteracting HU-induced replication stress (RS). Consistent with a role of REV3 in DNA replication, increased RPA staining was confined to HU-treated S-phase cells. Additionally, we found genes related to RS to be significantly enriched among the top hits of the synthetic sickness/lethality (SSL) screen further corroborating the importance of REV3 for DNA replication under conditions of RS.
authors.affiliation: Clinic of Oncology, University Hospital Zurich, 8044 Zurich, Switzerland.
authors.forename: Ilya N
authors.initials: IN
authors.lastname: Kotov
authors.affiliation: Department of Pulmonary Diseases, VU University Medical Center, 1081 HV Amsterdam, The Netherlands.
authors.forename: Ellen
authors.initials: E
authors.lastname: Siebring-van Olst
authors.affiliation: Clinic of Oncology, University Hospital Zurich, 8044 Zurich, Switzerland.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Knobel
authors.affiliation: Department of Medical Oncology, VU University Medical Center, 1081 HV Amsterdam, The Netherlands.
authors.forename: Ida H
authors.initials: IH
authors.lastname: van der Meulen-Muileman
authors.affiliation: Clinic of Oncology, University Hospital Zurich, 8044 Zurich, Switzerland.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Felley-Bosco
authors.affiliation: Department of Medical Oncology, VU University Medical Center, 1081 HV Amsterdam, The Netherlands.
authors.forename: Victor W
authors.initials: VW
authors.lastname: van Beusechem
authors.affiliation: Department of Pulmonary Diseases, VU University Medical Center, 1081 HV Amsterdam, The Netherlands.
authors.forename: Egbert F
authors.initials: EF
authors.lastname: Smit
authors.affiliation: Clinic of Oncology, University Hospital Zurich, 8044 Zurich, Switzerland.
authors.forename: Rolf A
authors.initials: RA
authors.lastname: Stahel
authors.affiliation: Clinic of Oncology, University Hospital Zurich, 8044 Zurich, Switzerland. Electronic address: thomas.marti@insel.ch.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Marti
mesh_terms: D045744:Cell Line, Tumor; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D034622:RNA Interference / Q000502:physiology; D012262:Ribonucleoside Diphosphate Reductase; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C475286:RRM1 protein, human; D012262:Ribonucleoside Diphosphate Reductase; D004259:DNA-Directed DNA Polymerase; C118285:REV3L protein, human
keywords: DNA damage; Polymerase zeta; REV3; Replication stress; Translesion synthesis
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