CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

Abstract

The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.

Molecular and physiological consequences of faulty eukaryotic ribonucleotide excision repair

“…Yet another scenario related to yeast Top1 activity at rNMP sites has been described: Following the first incision at the rNMP, Top1 can also cut on the DNA strand opposing the rNMP to create double-strand breaks (DSBs), which then rely on homology-directed repair (HDR) via Rad51 and Rad52 (Huang et al, 2015; Fig 2F). Consistently, RNase H2 loss in human cells results in synthetic lethality with the absence of either BRCA1 or BRCA2 HDR factors, highlighting the importance of HDR under conditions of rNMP accumulation (Zimmermann et al, 2018). Figure 2.…”

“…In yeast mutants, the S-phase checkpoint and the postreplicative repair pathway are constitutively activated, and accordingly, cells exhibit delayed cell cycle progression (Nick McElhinny et al, 2010a;Lazzaro et al, 2012;Williams et al, 2013;Zimmermann et al, 2018). In yeast mutants, the S-phase checkpoint and the postreplicative repair pathway are constitutively activated, and accordingly, cells exhibit delayed cell cycle progression (Nick McElhinny et al, 2010a;Lazzaro et al, 2012;Williams et al, 2013;Zimmermann et al, 2018).…”

“…The presence of rNMPs in a DNA template directly affects the processivity of DNA polymerization during semi-conservative replication. Therefore, the accumulation of rNMPs in genomic DNA likely induces replication stress and DNA damage signaling from yeast to human (Nick McElhinny et al, 2010a;Hiller et al, 2012;Lazzaro et al, 2012;Williams et al, 2013;Pizzi et al, 2015;Zimmermann et al, 2018) (discussed below). As of yet, there is no direct in vivo evidence that DNA polymerases are affected by stretches of multiple rNMPs.…”

“…Loss of any of its subunits renders the enzyme complex inactive (Jeong et al, 2004). Although this allele was originally constructed in yeast, it has since been recapitulated in human and mouse cells, resulting in a similar separationof-function phenotype (Pizzi et al, 2015;Uehara et al, 2018;Zimmermann et al, 2018). This point mutation within the substrate-interacting pocket completely abolishes activity of RNase H2 toward single rNMPs but retains approximately 40% of wildtype enzymatic activity toward longer rNMP stretches and R-loops (Chon et al, 2013).…”

“…While rNMP removal via topoisomerase had initially only been described in S. cerevisiae, recent work employing various RERdefective RNase H2-mutant human cell lines has demonstrated that also human TOP1 can recognize an unrepaired rNMP and incise to create a DNA nick with the potential to compromise genome stability (Zimmermann et al, 2018). Whether TOP1 activity indeed removes rNMPs from human DNA as it does in yeast remains to be tested.…”