SMARCAL1 ubiquitylation controls its association with RPA-coated ssDNA and promotes replication fork stability.

Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.

The mutational impact of Illudin S on human cells.

Illudin S (ILS) is a fungal sesquiterpene secondary metabolite with potent genotoxic and cytotoxic properties. Early genetic studies and more recent genome-wide CRISPR screens showed that Illudin-induced lesions are preferentially repaired by transcription-coupled nucleotide excision repair (TC-NER) with some contribution from post-replication repair pathways. In line with these results, Irofulven, a semi-synthetic ILS analog was recently shown to be particularly effective on cell lines and patient-derived xenografts with impaired NER (e.g. ERCC2/3 mutations), raising hope that ILS-derived molecules may soon enter the clinic. Despite the therapeutic potential of ILS and its analogs, we still lack a global understanding of their mutagenic potential. Here, we characterize the mutational signatures associated with chronic exposure to ILS in human cells. ILS treatment rapidly stalls DNA replication and transcription, leading to the activation of the replication stress response and the accumulation of DNA damage. Novel single and double base substitution signatures as well as a characteristic indel signature indicate that ILS treatment preferentially alkylates purine residues and induces oxidative stress, confirming prior in vitro data. Many mutation contexts exhibit a strong transcriptional strand bias, highlighting the contribution of TC-NER to the repair of ILS lesions. Finally, collateral mutations are also observed in response to ILS, suggesting a contribution of translesion synthesis pathways to ILS tolerance. Accordingly, ILS treatment led to the rapid recruitment of the Y-family DNA polymerase kappa onto chromatin, supporting its preferential use for ILS lesion bypass. Altogether, our work provides the first global assessment of the genomic impact of ILS, demonstrating the contribution of multiple DNA repair pathways to ILS resistance and mutagenicity.

Chronic treatment with ATR and CHK1 inhibitors does not substantially increase the mutational burden of human cells.

DNA replication stress (RS) entails the frequent slow down and arrest of replication forks by a variety of conditions that hinder accurate and processive genome duplication. Elevated RS leads to genome instability, replication catastrophe and eventually cell death. RS is particularly prevalent in cancer cells and its exacerbation to unsustainable levels by chemotherapeutic agents remains a cornerstone of cancer treatments. The adverse consequences of RS are normally prevented by the ATR and CHK1 checkpoint kinases that stabilize stressed forks, suppress origin firing and promote cell cycle arrest when replication is perturbed. Specific inhibitors of these kinases have been developed and shown to potentiate RS and cell death in multiple in vitro cancer settings. Ongoing clinical trials are now probing their efficacy against various cancer types, either as single agents or in combination with mainstay chemotherapeutics. Despite their promise as valuable additions to the anti-cancer pharmacopoeia, we still lack a genome-wide view of the potential mutagenicity of these new drugs. To investigate this question, we performed chronic long-term treatments of TP53-depleted human cancer cells with ATR and CHK1 inhibitors (ATRi, AZD6738/ceralasertib and CHK1i, MK8776/SCH-900776). ATR or CHK1 inhibition did not significantly increase the mutational burden of cells, nor generate specific mutational signatures. Indeed, no notable changes in the numbers of base substitutions, short insertions/deletions and larger scale rearrangements were observed despite induction of replication-associated DNA breaks during treatments. Interestingly, ATR inhibition did induce a slight increase in closely-spaced mutations, a feature previously attributed to translesion synthesis DNA polymerases. The results suggest that ATRi and CHK1i do not have substantial mutagenic effects in vitro when used as standalone agents.