Faculty Bibliography

One of the most central questions about the repair of a double-strand DNA break (DSB) concerns how the two free DNA ends are brought together - a step called synapsis. Using single-molecule FRET (smFRET), we show here that both Ku plus XRCC4:DNA ligase IV are necessary and sufficient to achieve a flexible synapsis of blunt DNA ends, whereas either alone is not. Addition of XLF causes a transition to a close synaptic state, and maximum efficiency of close synapsis is achieved within 20 min. The promotion of close synapsis by XLF indicates a role that is independent of a filament structure, with action focused at the very ends of each duplex. DNA-PKcs is not required for the formation of either the flexible or close synaptic states. This model explains in biochemical terms the evolutionarily central synaptic role of Ku, X4L4, and XLF in NHEJ for all eukaryotes.

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

Background: Nav1.5 is targeted to distinct subcellular microdomains of cardiomyocytes by the microtubule network, with sodium current being largest in the intercalated disc (ID) region. The microtubule plus-end tracking proteins End Binding 1 (EB1) and CLIP-associating protein 2 (CLASP2) are mainly located at the ID and regulate microtubule recruitment and stability. The small molecule SB216763 (SB2) acts on Glycogen synthase kinase 3 beta (GSK3beta) and is known to enhance the EB1-CLASP2 interaction, thereby increasing microtubule stability.
Objective(s): To investigate the effect of targeting EB1-CLASP2 on Nav1.5 localisation and sodium current density (INa) in subcellular microdomains.
Method(s): Patch clamp and Stochastic Optical Reconstruction Microscopy (STORM) imaging experiments were performed on human iPSC-derived cardiomyocytes (hiPSC-CMs) and freshly isolated murine ventricular cardiomyocytes.
Result(s): EB1 overexpression in hiPSC-CMs increased membrane Nav1.5 cluster density and consequently increased whole-cell INa and action potential (AP) upstroke velocity, without affecting INa kinetics or other AP parameters. Increased whole-cell INa was observed in murine cardiomyocytes after 2-4 hours of SB2 treatment (5micro M), while INa kinetics remained unaffected. Macropatch experiments revealed that SB2 specifically increased INa at the ID, while INa at the lateral membrane was unchanged. In contrast, SB2 did not affect INa or Nav1.5 cluster size or density in cardiomyocytes from mice Clasp2-deficient mice.
Conclusion(s): Targeting the plus-end tracking proteins EB1 and CLASP2 resulted in increased whole-cell peak INa in hiPSC-CMs and isolated murine cardiomyocytes. On the subcellular level, INa was specifically increased at the ID after pharmacologically enhancing the CLASP2-EB1 interaction by SB2. Treatment with SB2 in cardiomyocytes lacking CLASP2 did not affect INa or Nav1.5 distribution, demonstrating the central role for CLASP2 in the SB2-mediated effects. Thus, the microtubule-EB1-CLASP2 complex constitutes a promising target for modulating INa in a microdomain-specific manner.
Copyright

Multicolor single-molecule localization super-resolution microscopy has enabled visualization of ultrafine spatial organizations of molecular assemblies within cells. Despite many efforts, current approaches for distinguishing and quantifying such organizations remain limited, especially when these are contained within densely distributed super-resolution data. In theory, higher-order correlation such as the Triple-Correlation function is capable of obtaining the spatial configuration of individual molecular assemblies masked within seemingly discorded dense distributions. However, due to their enormous computational cost such analyses are impractical, even for high-end computers. Here, we developed a fast algorithm for Triple-Correlation analyses of high-content multiplexed super-resolution data. This algorithm computes the probability density of all geometric configurations formed by every triple-wise single-molecule localization from three different channels, circumventing impractical 4D Fourier Transforms of the entire megapixel image. This algorithm achieves 10²-folds enhancement in computational speed, allowing for high-throughput Triple-Correlation analyses and robust quantification of molecular complexes in multiplexed super-resolution microscopy.

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

DNA double-strand breaks (DSBs) are toxic DNA lesions, which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homologous recombination (HR) by contributing to the recruitment of the HR proteins BRCA1, BRCA2, and RAD51, without affecting DNA-end resection. In S/G2-phase cells, dilncRNAs pair to the resected DNA ends and form DNA:RNA hybrids, which are recognized by BRCA1. We also show that BRCA2 directly interacts with RNase H2, mediates its localization to DSBs in the S/G2 cell-cycle phase, and controls DNA:RNA hybrid levels at DSBs. These results demonstrate that regulated DNA:RNA hybrid levels at DSBs contribute to HR-mediated repair.

DNA replication stress is often defined by the slowing or stalling of replication fork progression leading to local or global DNA synthesis inhibition. Failure to resolve replication stress in a timely manner contribute towards cell cycle defects, genome instability and human disease; however, the mechanism for fork recovery remains poorly defined. Here we show that the translesion DNA polymerase (Pol) kappa, a DinB orthologue, has a unique role in both protecting and restarting stalled replication forks under conditions of nucleotide deprivation. Importantly, Pol kappa-mediated DNA synthesis during hydroxyurea (HU)-dependent fork restart is regulated by both the Fanconi Anemia (FA) pathway and PCNA polyubiquitination. Loss of Pol kappa prevents timely rescue of stalled replication forks, leading to replication-associated genomic instability, and a p53-dependent cell cycle defect. Taken together, our results identify a previously unanticipated role for Pol kappa in promoting DNA synthesis and replication stress recovery at sites of stalled forks.