Faculty Bibliography

The MRE11-RAD50-NBS1 (MRN) complex supports the synthesis of damage-induced long non-coding RNA (dilncRNA) by RNA polymerase II (RNAPII) from DNA double-strand breaks (DSBs) by an unknown mechanism. Here, we show that recombinant human MRN and native RNAPII are sufficient to reconstitute a minimal functional transcriptional apparatus at DSBs. MRN recruits and stabilizes RNAPII at DSBs. Unexpectedly, transcription is promoted independently from MRN nuclease activities. Rather, transcription depends on the ability of MRN to melt DNA ends, as shown by the use of MRN mutants and specific allosteric inhibitors. Single-molecule FRET assays with wild-type and mutant MRN show a tight correlation between the ability to melt DNA ends and to promote transcription. The addition of RPA enhances MRN-mediated transcription, and unpaired DNA ends allow MRN-independent transcription by RNAPII. These results support a model in which MRN generates single-strand DNA ends that favor the initiation of transcription by RNAPII.

DNA G-quadruplexes (G4s) are stable, non-canonical DNA secondary structures formed within guanine(G)-rich sequences. While extensively studied in vitro, evidence of the occurrence of G4s in vivo has only recently emerged. The formation of G4 structures may pose an obstacle for diverse DNA transactions including replication, which is linked to mutagenesis and genomic instability. A fundamental question in the field has been whether and how the formation of G4s is coupled to the progression of replication forks. This process has remained undefined largely due to the lack of experimental approaches capable of monitoring the presence of G4s and their association with the replication machinery in cells. Here, we describe a detailed multicolor single-molecule localization microscopy (SMLM) protocol for detecting nanoscale spatial-association of DNA G4s with the cellular replisome complex. This method offers a unique platform for visualizing the mechanisms of G4 formation at the molecular level, as well as addressing key biological questions as to the functional roles of these structures in the maintenance of genome integrity.

Single-molecule super-resolution microscopy (SRM) combines single-molecule detection with spatial resolutions tenfold improved over conventional confocal microscopy. These two key advantages make it possible to visualize individual DNA replication and damage events within the cellular context of fixed cells. This in turn engenders the ability to decipher variations between individual replicative and damage species within a single nucleus, elucidating different subpopulations of stress and repair events. Here, we describe the protocol for combining SRM with novel labeling and damage assays to characterize DNA double-strand break (DSB) induction at stressed replication forks (RFs) and subsequent repair by homologous recombination (HR). These assays enable spatiotemporal mapping of DNA damage response and repair proteins to establish their in vivo function and interactions, as well as detailed characterization of specific dysfunctions in HR caused by drugs or mutations of interest.

Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). Super-resolution microscopy has made it possible to directly observe replication stress and DNA damage inside cells, however new approaches to sample preparation and analysis are required. Here we develop and apply multicolor single molecule microscopy to visualize individual replication forks under mild stress from the trapping of Topoisomerase I cleavage complexes, a damage induction which closely mimics endogenous replicative stress. We observe RAD51 and RAD52, alongside RECQ1, as the first responder proteins to stalled but unbroken forks, whereas Ku and MRE11 are initially recruited to seDSBs. By implementing novel super-resolution imaging assays, we are thus able to discern closely related replication fork stress motifs and their repair pathways.

Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism of any type of DNA double-strand break (DSB) during most of the cell cycle and is essential for the development of antigen receptors. Defects in NHEJ result in sensitivity to ionizing radiation and loss of lymphocytes. The most critical step of NHEJ is synapsis, or the juxtaposition of the two DNA ends of a DSB, because all subsequent steps rely on it. Recent findings show that, like the end processing step, synapsis can be achieved through several mechanisms. In this Review, we first discuss repair pathway choice between NHEJ and other DSB repair pathways. We then integrate recent insights into the mechanisms of NHEJ synapsis with updates on other steps of NHEJ, such as DNA end processing and ligation. Finally, we discuss NHEJ-related human diseases, including inherited disorders and neoplasia, which arise from rare failures at different NHEJ steps.

Numerous anti-cancer drugs perturb thymidylate biosynthesis and lead to genomic uracil incorporation contributing to their antiproliferative effect. Still, it is not yet characterized if uracil incorporations have any positional preference. Here, we aimed to uncover genome-wide alterations in uracil pattern upon drug treatments in human cancer cell line models derived from HCT116. We developed a straightforward U-DNA sequencing method (U-DNA-Seq) that was combined with in situ super-resolution imaging. Using a novel robust analysis pipeline, we found broad regions with elevated probability of uracil occurrence both in treated and non-treated cells. Correlation with chromatin markers and other genomic features shows that non-treated cells possess uracil in the late replicating constitutive heterochromatic regions, while drug treatment induced a shift of incorporated uracil towards segments that are normally more active/functional. Data were corroborated by colocalization studies via dSTORM microscopy. This approach can be applied to study the dynamic spatio-temporal nature of genomic uracil.

Background - Mutations in the gene encoding the sodium channel Na_v1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Na_v1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Na_v1.5 localization and function remain insufficiently characterized. Methods - We used novel single-molecule localization microscopy (SMLM) and computational modeling to define nanoscale features of Na_v1.5 localization and distribution at the lateral membrane (LM), the LM groove, and T-tubules (TT) in cardiomyocytes from wild-type (N = 3), dystrophin-deficient (mdx; N = 3) mice, and mice expressing C-terminally truncated Na_v1.5 (ΔSIV; N = 3). We moreover assessed TT sodium current by recording whole-cell sodium currents in control (N = 5) and detubulated (N = 5) wild-type cardiomyocytes. Results - We show that Na_v1.5 organizes as distinct clusters in the groove and T-tubules which density, distribution, and organization partially depend on SIV and dystrophin. We found that overall reduction in Na_v1.5 expression in mdx and ΔSIV cells results in a non-uniform re-distribution with Na_v1.5 being specifically reduced at the groove of ΔSIV and increased in T-tubules of mdx cardiomyocytes. A TT sodium current could however not be demonstrated. Conclusions - Na_v1.5 mutations may site-specifically affect Na_v1.5 localization and distribution at the lateral membrane and T-tubules, depending on site-specific interacting proteins. Future research efforts should elucidate the functional consequences of this redistribution.

FANCJ/BRIP1 is an iron-sulfur (FeS) cluster-binding DNA helicase involved in DNA inter-strand cross-link (ICL) repair and G-quadruplex (G4) metabolism. Mutations in FANCJ are associated with Fanconi anemia and an increased risk for developing breast and ovarian cancer. Several cancer-associated mutations are located in the FeS domain of FANCJ, but how they affect FeS cluster binding and/or FANCJ activity has remained mostly unclear. Here we show that the FeS cluster is indispensable for FANCJ's ability to unwind DNA substrates in vitro and to provide cellular resistance to agents that induce ICLs. Moreover, we find that FANCJ requires an intact FeS cluster for its ability to unfold G4 structures on the DNA template in a primer extension assay with the lagging-strand DNA polymerase delta. Surprisingly, however, FANCJ variants that are unable to bind an FeS cluster and to unwind DNA in vitro can partially suppress the formation of replisome-associated G4 structures that we observe in a FANCJ knock-out cell line. This may suggest a partially retained cellular activity of FANCJ variants with alterations in the FeS domain. On the other hand, FANCJ knock-out cells expressing FeS cluster-deficient variants display a similar-enhanced-sensitivity towards pyridostatin (PDS) and CX-5461, two agents that stabilise G4 structures, as FANCJ knock-out cells. Mutations in FANCJ that abolish FeS cluster binding may hence be predictive of an increased cellular sensitivity towards G4-stabilising agents.

mRNAs enriched in membraneless condensates provide functional compartmentalization within cells. The mechanisms that recruit transcripts to condensates are under intense study; however, how mRNAs organize once they reach a granule remains poorly understood. Here, we report on a self-sorting mechanism by which multiple mRNAs derived from the same gene assemble into discrete homotypic clusters. We demonstrate that in vivo mRNA localization to granules and self-assembly within granules are governed by different mRNA features: localization is encoded by specific RNA regions, whereas self-assembly involves the entire mRNA, does not involve sequence-specific, ordered intermolecular RNA:RNA interactions, and is thus RNA sequence independent. We propose that the ability of mRNAs to self-sort into homotypic assemblies is an inherent property of an messenger ribonucleoprotein (mRNP) that is augmented under conditions that increase RNA concentration, such as upon enrichment in RNA-protein granules, a process that appears conserved in diverse cellular contexts and organisms.

The efficient maintenance of genome integrity in the face of cellular stress is vital to protect against human diseases such as cancer. DNA replication, chromatin dynamics, cellular signaling, nuclear architecture, cell cycle checkpoints, and other cellular activities contribute to the delicate spatiotemporal control that cells utilize to regulate and maintain genome stability. This perspective will highlight DNA double-strand break (DSB) repair pathways in human cells, how DNA repair failures can lead to human disease, and how PARP inhibitors have emerged as a novel clinical therapy to treat homologous recombination-deficient tumors. We briefly discuss how failures in DNA repair produce a permissive genetic environment in which preneoplastic cells evolve to reach their full tumorigenic potential. Finally, we conclude that an in-depth understanding of DNA DSB repair pathways in human cells will lead to novel therapeutic strategies to treat cancer and potentially other human diseases.