Faculty Bibliography

Schlafen family member 11 (SLFN11) expression sensitizes cells to a spectrum of DNA-damaging chemotherapies. Previous studies have shown that SLFN11 is recruited to stalled replication forks in response to replication stress; however, the role of SLFN11 at stressed replication forks remains unclear. Using single-molecule DNA fiber analysis and super-resolution microscopy to interrogate the dynamics of individual replication forks, we show that SLFN11 acts upon stalled replication forks to suppress efficient fork restart. In the absence of SLFN11 expression, fork restart proceeds through a pathway involving the ubiquitin ligase RFWD3 and the DNA primase-polymerase PRIMPOL to facilitate gapped DNA synthesis, thereby ensuring that cells do not accumulate replication-associated DNA damage. SLFN11 antagonizes this pathway by disrupting recruitment of RFWD3 and PRIMPOL to stalled forks in a manner dependent on a functional ATPase domain and persistent fork localization, but not on tRNA hydrolysis or ssDNA binding. Collectively, our results provide a mechanistic basis for how SLFN11 can counteract DNA damage tolerance by suppressing the RFWD3-PRIMPOL fork restart pathway.

In eukaryotes, each ribosomal subunit includes a ribosomal protein (RP) that is encoded as a fusion protein with ubiquitin (Ub). In yeast, each Ub-RP fusion requires processing by deubiquitylating enzymes (DUBs) to generate ribosome assembly-competent RPs and contribute to the cellular Ub pool. However, how Ub-RP fusions are processed by DUBs in human cells remains unclear. Here, we discovered that Ub-RPs are substrates of the Ub-fusion degradation (UFD) pathway in human cells via lysine 29 and 48 (K29/K48)-specific ubiquitylation and proteasomal degradation. We identified a pool of DUBs that catalytically process Ub-RPs, as well as DUBs that physically occlude Ub-RP interaction with UFD pathway Ub E3 ligases to prevent their degradation in a non-catalytic manner. Our results suggest that DUBs both process and stabilize Ub-RPs, whereas the UFD pathway regulates levels of Ub-RPs that cannot be fully processed by DUBs to fine-tune protein homeostasis.

The cellular networks that maintain genome stability encompass numerous pathways involved in all aspects of nucleic acid metabolism. Through bioinformatic analysis, we identified the Zinc Finger CCCH-Type Containing 4 protein (ZC3H4), a suppressor of noncoding RNA (ncRNA) production, as a pivotal player in this system. Experimentally, ZC3H4 deficiency led to increased DNA damage, abnormal mitosis, and cellular senescence. Biochemical analysis and super-resolution microscopy revealed that the loss of ZC3H4 increased replication stress (RS)-a major driver of genome instability-by inducing a hypertranscription state that promoted R loop formation and transcription-replication conflicts (TRCs), both of which drive RS. Further bioinformatic analysis demonstrated that ZC3H4 preferentially binds to genomic regions prone to TRCs and R loops, where it suppresses ncRNA bursts, functioning as part of the Restrictor complex. Our findings identify ZC3H4 as a crucial factor in maintaining genome integrity, strategically positioned at the critical intersection of DNA and RNA synthesis.

The tumour-suppressor protein BRCA2 has a central role in homology-directed DNA repair by enhancing the formation of RAD51 filaments on resected single-stranded DNA generated at double-stranded DNA breaks and stimulating RAD51 activity^1,2. Individuals with BRCA2 mutations are predisposed to cancer; however, BRCA2-deficient tumours are often responsive to targeted therapy with PARP inhibitors (PARPi)^3-6. The mechanism by which BRCA2 deficiency renders cells sensitive to PARPi but with minimal toxicity in cells heterozygous for BRCA2 mutations remains unclear. Here we identify a previously unknown role of BRCA2 that is directly linked to the effect of PARP1 inhibition. Using biochemical and single-molecule approaches, we demonstrate that PARPi-mediated PARP1 retention on a resected DNA substrate interferes with RAD51 filament stability and impairs RAD51-mediated DNA strand exchange. Full-length BRCA2 protects RAD51 filaments and counteracts the instability conferred by PARPi-mediated retention by preventing the binding of PARP1 to DNA. Extending these findings to a cellular context, we use quantitative single-molecule localization microscopy to show that BRCA2 prevents PARPi-induced PARP1 retention at homologous-recombination repair sites. By contrast, BRCA2-deficient cells exhibit increased PARP1 retention at these lesions in response to PARPi. These results provide mechanistic insights into the role of BRCA2 in maintaining RAD51 stability and protecting homologous-recombination repair sites by mitigating PARPi-mediated PARP1 retention.

Mammalian genomes replicate in a cell-type-specific order during the S phase, correlated to transcriptional activity, histone modifications, and chromatin structure. The causal relationships between these features and DNA replication timing (RT), especially during cell fate changes, are largely unknown. Using machine learning, we quantify 21 chromatin features predicting local RT and RT changes during differentiation in embryonic stem cells (ESCs). About one-third of the genome shows RT changes during differentiation. Chromatin features accurately predict both steady-state RT and RT changes. Histone H3 lysine 4 monomethylation (H3K4me1), catalyzed by KMT2C and KMT2D (KMT2C/D), emerges as a top predictor. Loss of KMT2C/D or their enzymatic activities impairs RT changes during differentiation. This correlates with local H3K4me1 loss and reduced replication origin firing, while transcription remains largely unaffected. Our findings reveal KMT2C/D-dependent H3K4me1 as a key regulator of RT and replication initiation, a role that likely impacts diseases associated with KMT2C/D mutations.

Cancer cells often experience nutrient-limiting conditions because of their robust proliferation and inadequate tumour vasculature, which results in metabolic adaptation to sustain proliferation. Most cancer cells rapidly consume glucose, which is severely reduced in the nutrient-scarce tumour microenvironment. In CRISPR-based genetic screens to identify metabolic pathways influenced by glucose restriction, we find that tumour-relevant glucose concentrations (low glucose) protect cancer cells from inhibition of de novo pyrimidine biosynthesis, a pathway that is frequently targeted by chemotherapy. We identify two mechanisms to explain this result, which is observed broadly across cancer types. First, low glucose limits uridine-5-diphosphate-glucose synthesis, preserving pyrimidine nucleotide availability and thereby prolonging the time to replication fork stalling. Second, low glucose directly modulates apoptosis downstream of replication fork stalling by suppressing BAK activation and subsequent cytochrome c release, key events that activate caspase-9-dependent mitochondrial apoptosis. These results indicate that the low glucose levels frequently observed in tumours may limit the efficacy of specific chemotherapeutic agents, highlighting the importance of considering the effects of the tumour nutrient environment on cancer therapy.

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

Transcription and replication both require large macromolecular complexes to act on a DNA template, yet these machineries cannot simultaneously act on the same DNA sequence. Conflicts between the replication and transcription machineries (transcription-replication conflicts, or TRCs) are widespread in both prokaryotes and eukaryotes and have the capacity to both cause DNA damage and compromise complete, faithful replication of the genome. This review will highlight recent studies investigating the genomic locations of TRCs and the mechanisms by which they may be prevented, mitigated, or resolved. We address work from both model organisms and mammalian systems but predominantly focus on multicellular eukaryotes owing to the additional complexities inherent in the coordination of replication and transcription in the context of cell type-specific gene expression and higher-order chromatin organization.

Classic experiments with peripheral sympathetic neurons established an absolute dependence upon NGF for survival. A forgotten problem is how these neurons become resistant to deprivation of trophic factors. The question is whether and how neurons can survive in the absence of trophic support. However, the mechanism is not understood how neurons switch their phenotype to lose their dependence on trophic factors, such as NGF and BDNF. Here, we approach the problem by considering the requirements for trophic support of peripheral sympathetic neurons and hippocampal neurons from the central nervous system. We developed cellular assays to assess trophic factor dependency for sympathetic and hippocampal neurons and identified factors that rescue neurons in the absence of trophic support. They include enhanced expression of a subunit of the NGF receptor (Neurotrophin Receptor Homolog, NRH) in sympathetic neurons and an increase of the expression of the glucocorticoid receptor in hippocampal neurons. The results are significant since levels and activity of trophic factors are responsible for many neuropsychiatric conditions. Resistance of neurons to trophic factor deprivation may be relevant to the underlying basis of longevity, as well as an important element in preventing neurodegeneration.

The deubiquitinase USP1 is a critical regulator of genome integrity through the deubiquitylation of Fanconi Anemia proteins and the DNA replication processivity factor, proliferating cell nuclear antigen (PCNA). Uniquely, following UV irradiation, USP1 self-inactivates through autocleavage, which enables its own degradation and in turn, upregulates PCNA monoubiquitylation. However, the functional role for this autocleavage event during physiological conditions remains elusive. Herein, we discover that cells harboring an autocleavage-defective USP1 mutant, while still able to robustly deubiquitylate PCNA, experience more replication fork-stalling and premature fork termination events. Using super-resolution microscopy and live-cell single-molecule tracking, we show that these defects are related to the inability of this USP1 mutant to be properly recycled from sites of active DNA synthesis, resulting in replication-associated lesions. Furthermore, we find that the removal of USP1 molecules from DNA is facilitated by the DNA-dependent metalloprotease Spartan to counteract the cytotoxicity caused by "USP1-trapping". We propose a utility of USP1 inhibitors in cancer therapy based on their ability to induce USP1-trapping lesions and consequent replication stress and genomic instability in cancer cells, similar to how non-covalent DNA-protein crosslinks cause cytotoxicity by imposing steric hindrances upon proteins involved in DNA transactions.