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.

Rscreenorm: normalization of CRISPR and siRNA screen data for more reproducible hit selection

“…We applied rscreenorm to genome-wide siRNA screens [ 14 – 16 ] data of 7 human cell lines (see Additional file 1 ). After centering each replicate around the negative controls’ median, we could see that (log2-) measured viabilities displayed considerable variability between cell lines: the viability range width represented by the difference between negative and positive controls’ medians varies between 0.7 for one replicate of cell line VU-SCC-120 and 5.1 for replicate 3 of cell line 786-O (Additional file 1 : Figure S5).…”

A Synthetic Lethal Screen Identifies DNA Repair Pathways that Sensitize Cancer Cells to Combined ATR Inhibition and Cisplatin Treatments

“…Pol ζ is viewed as a master regulator in TLS, as Pol ζ and REV1 are required to complete repair initiated by several other TLS polymerases [ 40 ]. In addition to its role in cisplatin resistance, REV3 is also required for maintaining viability after replication fork stalling and for preventing expression of common fragile sites [ 46 , 47 ]. Loss of REV3 also causes persistent DNA damage and an increase in γH2AX [ 48 ].…”

Inhibition of mutagenic translesion synthesis: A possible strategy for improving chemotherapy?

“…Although a compelling idea, TLS synthetic partners are largely unknown. However, a whole genome siRNA library screen in A549 lung cancer cells identified one gene RRMI—the large subunit of ribonucleotide reductase that confers a synthetic lethal interaction with REV3 [ 113 ]. In another lung cancer cell line and in breast cancer cells, ataxia-telangiectasia and Rad3 related inhibition was found to synthetically enhance lethality in cisplatin-treated REV3-deficient cells [ 114 ].…”