Dicer prevents genome instability in response to replication stress

Dicer, an endoribonuclease best-known for its role in microRNA biogenesis and RNA interference pathway, has been shown to play a role in the DNA damage response and repair of double-stranded DNA breaks (DSBs) in mammalian cells. However, it remains unknown whether Dicer is also important to preserve genome integrity upon replication stress. To address this question, we focused our study on common fragile sites (CFSs), which are susceptible to breakage after replication stress. We show that inhibition of the Dicer pathway leads to an increase in CFS expression upon induction of replication stress and to an accumulation of 53BP1 nuclear bodies, indicating transmission of replication-associated damage. We also show that in absence of a functional Dicer or Drosha, the assembly into nuclear foci of the Fanconi anemia (FA) protein FANCD2 and of the replication and checkpoint factor TopBP1 in response to replication stress is impaired, and the activation of the S-phase checkpoint is defective. Based on these results, we propose that Dicer pre-vents genomic instability after replication stress, by allowing the proper recruitment to stalled forks of proteins that are necessary to maintain replication fork stability and activate the S-phase checkpoint, thus limiting cells from proceeding into mitosis with under-replicated DNA

Integrating DNA damage repair with the cell cycle

Abstract DNA is labile and constantly subject to damage. In addition to external mutagens, DNA is continuously damaged by the aqueous environment, cellular metabolites and is prone to strand breakage during replication. Cell duplication is orchestrated by the cell division cycle and specific DNA structures are processed differently depending on where in the cell cycle they are detected. This is often because a specific structure is physiological in one context, for example during DNA replication, while indicating a potentially pathological event in another, such as interphase or mitosis. Thus, contextualising the biochemical entity with respect to cell cycle progression provides information necessary to appropriately regulate DNA processing activities. We review the links between DNA repair and cell cycle context, drawing together recent advances

Context-dependent pro- and anti-resection roles of ZKSCAN3 in the regulation of fork processing during replication stress

Uncontrolled resection of replication forks under stress can cause genomic instability and influence cancer formation. Extensive fork resection has also been implicated in the chemosensitivity of BReast CAncer gene BRCA-deficient cancers. However, how fork resection is controlled in different genetic contexts and how it affects chromosomal stability and cell survival remains incompletely understood. Here, we report a novel function of the transcription repressor ZKSCAN3 in fork protection and chromosomal stability maintenance under replication stress. We show disruption of ZKSCAN3 function causes excessive resection of replication forks by the exonuclease Exo1 and homologous DNA recombination/repair protein Mre11 following fork reversal. Interestingly, in BRCA1-deficient cells, we found ZKSCAN3 actually promotes fork resection upon replication stress. We demonstrate these anti- and pro-resection roles of ZKSCAN3, consisting of a SCAN box, Kruppel-associated box, and zinc finger domain, are mediated by its SCAN box domain and do not require the Kruppel-associated box or zinc finger domains, suggesting that the transcriptional function of ZKSCAN3 is not involved. Furthermore, despite the severe impact on fork structure and chromosomal stability, depletion of ZKSCAN3 did not affect the short-term survival of BRCA1-proficient or BRCA1-deficient cells after treatment with cancer drugs hydroxyurea, PARPi, or cisplatin. Our findings reveal a unique relationship between ZKSCAN3 and BRCA1 in fork protection and add to our understanding of the relationships between replication fork protection, chromosomal instability, and chemosensitivity

A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells

BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18-UBC13-PALB2-RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells

Abstract 803: Identifying a RAD18/UBC13-dependent mechanism of replication fork recovery to modulate chemoresponse in BRCA1-deficient cancers

Abstract Mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 are associated with an increased lifetime risk of breast and ovarian cancers. While the BRCA proteins play a well-established role in double-stranded DNA break repair, recent studies have revealed an emerging role of BRCA1/2 in replication stress response. While replication forks are extensively degraded by nucleases in BRCA-deficient cancer cells, activation of specialized fork recovery mechanisms enables resumption of DNA synthesis and promotes cell survival. My project aims to determine this fork recovery mechanism in BRCA1-deficient cells and to identify potential recovery factors that can be targeted to improve chemotherapeutic response in BRCA1-mutated breast and ovarian cancers. To monitor perturbations in replication fork dynamics on a genome-wide scale, we utilize a DNA fiber assay technique measuring rates of fork recovery and replication fork degradation. In parallel, electron microscopy analysis allows direct visualization of replication fork intermediates. Cell survival assays are employed to test how loss of fork recovery factors impacts cell proliferation and chemotherapeutic response in BRCA1-deficient cells. Our results reveal that RAD18 and UBC13, which catalyze ubiquitination of Proliferating Cellular Nuclear Antigen (PCNA), promote fork recovery in BRCA1-deficient, but not BRCA2-deficient, cancer cells. Previous work has also shown that PCNA polyubiquitination by UBC13 is important for reversed fork formation in BRCA-proficient cells. However, our findings show that extensive degradation of reversed fork substrates still occurs in BRCA1-deficient cells lacking RAD18 or UBC13, indicating that PCNA polyubiquitination is not essential for fork reversal in this genetic background. In addition, loss of RAD18 in BRCA1-deficient cells significantly slows cell proliferation, and UBC13 inhibition further sensitizes cells lacking BRCA1 to the replication stress inducer Hydroxyurea (HU). Based on our findings, we hypothesize that RAD18, UBC13, and PCNA ubiquitination may represent novel targets to improve chemoresponse in BRCA1-deficient cancers that rely on fork recovery mechanisms for survival. Citation Format: Emily Cybulla, Jessica Jackson, Stephanie Tirman, Annabel Quinet, Delphine Lemacon, Alessandro Vindigni. Identifying a RAD18/UBC13-dependent mechanism of replication fork recovery to modulate chemoresponse in BRCA1-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 803.</jats:p