Whole genome RNAi screens reveal a critical role of REV3 in coping with replication stress. Issue 8 (22nd July 2014)

• Record Type: Journal Article
• Authors: Kotov, Ilya N.; Siebring-van Olst, Ellen; Knobel, Philip A.; van der Meulen-Muileman, Ida H.; Felley-Bosco, Emanuela; van Beusechem, Victor W.; Smit, Egbert F.; Stahel, Rolf A.; Marti, Thomas M.
• Is Part Of: Molecular oncology. Volume 8:Issue 8(2014:Dec.)
• Page Start: 1747
• Page End: 1759
• Publication Date: 2014-07-22
• Subjects: REV3 -- Polymerase zeta -- Replication stress -- Translesion synthesis -- DNA damage; Cancer -- Molecular aspects -- Periodicals; 616.994005
• DOI: 10.1016/j.molonc.2014.07.008
• Languages: English
• ISSNs: 1574-7891

Abstract

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. Highlights: Whole‐genome RNAi synthetic sickness/lethality screens were performed. We identified synthetic sickness/lethality interaction of RRM1 with REV3. HU and iREV3 treatments synergistically induce single‐stranded DNA in S‐phase. This increase is not accompanied by accumulation of DNA damage. Our findings indicate that REV3 plays a role in coping with DNA replication stress.