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Loss of Nuclear Envelope Integrity and Increased Oxidant Production Cause DNA Damage in Adult Hearts Deficient in PKP2: A Molecular Substrate of ARVC

Pérez-Hernández, Marta; van Opbergen, Chantal J M; Bagwan, Navratan; Rasmus Vissing, Christoffer; Marrón-Liñares, Grecia M; Zhang, Mingliang; Torres Vega, Estefania; Sorrentino, Andrea; Drici, Lylia; Sulek, Karolina; Zhai, Ruxu; Hansen, Finn B; Hørby Christensen, Alex; Boesgaard, Søren; Gustafsson, Finn; Rossing, Kasper; Small, Eric M; Davies, Michael J; Rothenberg, Eli; Sato, Priscila; Cerrone, Marina; Jensen, Thomas Hartvig Lindkær; Qvortrup, Klaus; Bundgaard, Henning; Delmar, Mario; Lundby, Alicia
BACKGROUND:gene, which encodes the PKP2 protein (plakophilin-2). METHODS:studied at a time of preserved left ventricular ejection fraction and in human induced pluripotent stem cell-derived PKP2-deficient myocytes. RESULTS: CONCLUSIONS:
PMID: 35959657
ISSN: 1524-4539
CID: 5287322

Preserved cardiac performance and adrenergic response in a rabbit model with decreased ryanodine receptor 2 expression

Zheng, Jingjing; Dooge, Holly C; Pérez-Hernández, Marta; Zhao, Yan-Ting; Chen, Xi; Hernandez, Jonathan J; Valdivia, Carmen R; Palomeque, Julieta; Rothenberg, Eli; Delmar, Mario; Valdivia, Héctor H; Alvarado, Francisco J
Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca2+ required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca2+-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca2+ handling in the heart. We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO+/-) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO+/- myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca2+ spark frequency and amplitude; (3) slower rate of Ca2+ release and mild but significant desynchronization of the Ca2+ transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A. Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca2+ release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus.
PMID: 35413295
ISSN: 1095-8584
CID: 5201912

USP1-trapping lesions as a source of DNA replication stress and genomic instability

Coleman, Kate E; Yin, Yandong; Lui, Sarah Kit Leng; Keegan, Sarah; Fenyo, David; Smith, Duncan J; Rothenberg, Eli; Huang, Tony T
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.
PMID: 35365626
ISSN: 2041-1723
CID: 5201472

V(D)J Recombination: Recent Insights in Formation of the Recombinase Complex and Recruitment of DNA Repair Machinery

Christie, Shaun M; Fijen, Carel; Rothenberg, Eli
V(D)J recombination is an essential mechanism of the adaptive immune system, producing a diverse set of antigen receptors in developing lymphocytes via regulated double strand DNA break and subsequent repair. DNA cleavage is initiated by the recombinase complex, consisting of lymphocyte specific proteins RAG1 and RAG2, while the repair phase is completed by classical non-homologous end joining (NHEJ). Many of the individual steps of this process have been well described and new research has increased the scale to understand the mechanisms of initiation and intermediate stages of the pathway. In this review we discuss 1) the regulatory functions of RAGs, 2) recruitment of RAGs to the site of recombination and formation of a paired complex, 3) the transition from a post-cleavage complex containing RAGs and cleaved DNA ends to the NHEJ repair phase, and 4) the potential redundant roles of certain factors in repairing the break. Regulatory (non-core) domains of RAGs are not necessary for catalytic activity, but likely influence recruitment and stabilization through interaction with modified histones and conformational changes. To form long range paired complexes, recent studies have found evidence in support of large scale chromosomal contraction through various factors to utilize diverse gene segments. Following the paired cleavage event, four broken DNA ends must now make a regulated transition to the repair phase, which can be controlled by dynamic conformational changes and post-translational modification of the factors involved. Additionally, we examine the overlapping roles of certain NHEJ factors which allows for prevention of genomic instability due to incomplete repair in the absence of one, but are lethal in combined knockouts. To conclude, we focus on the importance of understanding the detail of these processes in regards to off-target recombination or deficiency-mediated clinical manifestations.
PMID: 35573672
ISSN: 2296-634x
CID: 5284182

A basal-level activity of ATR links replication fork surveillance and stress response

Yin, Yandong; Lee, Wei Ting Chelsea; Gupta, Dipika; Xue, Huijun; Tonzi, Peter; Borowiec, James A; Huang, Tony T; Modesti, Mauro; Rothenberg, Eli
Mammalian cells use diverse pathways to prevent deleterious consequences during DNA replication, yet the mechanism by which cells survey individual replisomes to detect spontaneous replication impediments at the basal level, and their accumulation during replication stress, remain undefined. Here, we used single-molecule localization microscopy coupled with high-order-correlation image-mining algorithms to quantify the composition of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a basal-level activity of ATR that monitors and regulates the amounts of RPA at forks during normal replication. Replication-stress amplifies the basal activity through the increased volume of ATR-RPA interaction and diffusion-driven enrichment of ATR at forks. This localized crowding of ATR enhances its collision probability, stimulating the activation of its replication-stress response. Finally, we provide a computational model describing how the basal activity of ATR is amplified to produce its canonical replication stress response.
PMID: 34473946
ISSN: 1097-4164
CID: 5000152

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency

Cong, Ke; Peng, Min; Kousholt, Arne Nedergaard; Lee, Wei Ting C; Lee, Silviana; Nayak, Sumeet; Krais, John; VanderVere-Carozza, Pamela S; Pawelczak, Katherine S; Calvo, Jennifer; Panzarino, Nicholas J; Jonkers, Jos; Johnson, Neil; Turchi, John J; Rothenberg, Eli; Cantor, Sharon B
Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.
PMID: 34216544
ISSN: 1097-4164
CID: 4932752

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency

Cong, Ke; Peng, Min; Kousholt, Arne Nedergaard; Lee, Wei Ting C; Lee, Silviana; Nayak, Sumeet; Krais, John; VanderVere-Carozza, Pamela S; Pawelczak, Katherine S; Calvo, Jennifer; Panzarino, Nicholas J; Turchi, John J; Johnson, Neil; Jonkers, Jos; Rothenberg, Eli; Cantor, Sharon B
PMID: 34358459
ISSN: 1097-4164
CID: 5000142

Targeting the Microtubule EB1-CLASP2 Complex Modulates NaV1.5 at Intercalated Discs

Marchal, Gerard A; Jouni, Mariam; Chiang, David Y; Pérez-Hernández Duran, Marta; Podliesna, Svitlana; Yu, Nuo; Casini, Simona; Potet, Franck; Veerman, Christiaan C; Klerk, Mischa; Lodder, Elisabeth M; Mengarelli, Isabella; Guan, Kaomei; Vanoye, Carlos G; Rothenberg, Eli; Charpentier, Flavien; Redon, Richard; George, Alfred; Verkerk, Arie O; Bezzina, Connie R; MacRae, Calum A; Burridge, Paul; Delmar, Mario; Galjart, Niels J; Portero, Vincent; Remme, Carol Ann
Rationale: Loss-of-function of the cardiac sodium channel NaV1.5 causes conduction slowing and arrhythmias. NaV1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current (INa) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific INa reduction, but the mechanisms underlying microdomain-specific targeting of NaV1.5 remain largely unknown. Objective: To investigate the role of the microtubule (MT) plus-end tracking proteins end binding protein 1 (EB1) and CLIP-associated protein 2 (CLASP2) in mediating NaV1.5 trafficking and subcellular distribution in cardiomyocytes.Methods and Results: EB1 overexpression in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) resulted in enhanced whole-cell INa, increased action potential (AP) upstroke velocity (Vmax), and enhanced NaV1.5 localization at the plasma membrane as detected by multi-color stochastic optical reconstruction microscopy (STORM). Fluorescence recovery after photobleaching (FRAP) experiments in HEK293A cells demonstrated that EB1 overexpression promoted NaV1.5 forward trafficking. Knockout of MAPRE1 in hiPSC-CMs led to reduced whole-cell INa, decreased Vmax and AP duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish (mapre1b) resulted in decreased ventricular conduction velocity and Vmax as well as increased APD. STORM imaging and macropatch INa measurements showed that subacute treatment (2-3 hours) with SB216763 (SB2), a GSK3β inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3β localization and increased NaV1.5 and INa preferentially at the ID region of wild type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell INa or NaV1.5 localization in cardiomyocytes from Clasp2-deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of NaV1.5 at the ID. Conclusions: Our findings demonstrate the modulatory effect of the MT plus-end tracking protein EB1 on NaV1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of INa within the ID region of cardiomyocytes.
PMID: 34092082
ISSN: 1524-4571
CID: 4899502

The evolving complexity of DNA damage foci: RNA, condensates and chromatin in DNA double-strand break repair

Fijen, Carel; Rothenberg, Eli
Formation of biomolecular condensates is increasingly recognized as a mechanism employed by cells to deal with stress and to optimize enzymatic reactions. Recent studies have characterized several DNA repair foci as phase-separated condensates, behaving like liquid droplets. Concomitantly, the apparent importance of long non-coding RNAs and RNA-binding proteins for the repair of double-strand breaks has raised many questions about their exact contribution to the repair process. Here we discuss how RNA molecules can participate in condensate formation and how RNA-binding proteins can act as molecular scaffolds. We furthermore summarize our current knowledge about how properties of condensates can influence the choice of repair pathway (homologous recombination or non-homologous end joining) and identify the open questions in this field of emerging importance.
PMID: 34256335
ISSN: 1568-7856
CID: 4938412

Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling

Lee, Wei Ting C; Yin, Yandong; Morten, Michael J; Tonzi, Peter; Gwo, Pam Pam; Odermatt, Diana C; Modesti, Mauro; Cantor, Sharon B; Gari, Kerstin; Huang, Tony T; Rothenberg, Eli
Guanine-rich DNA sequences occur throughout the human genome and can transiently form G-quadruplex (G4) structures that may obstruct DNA replication, leading to genomic instability. Here, we apply multi-color single-molecule localization microscopy (SMLM) coupled with robust data-mining algorithms to quantitatively visualize replication fork (RF)-coupled formation and spatial-association of endogenous G4s. Using this data, we investigate the effects of G4s on replisome dynamics and organization. We show that a small fraction of active replication forks spontaneously form G4s at newly unwound DNA immediately behind the MCM helicase and before nascent DNA synthesis. These G4s locally perturb replisome dynamics and organization by reducing DNA synthesis and limiting the binding of the single-strand DNA-binding protein RPA. We find that the resolution of RF-coupled G4s is mediated by an interplay between RPA and the FANCJ helicase. FANCJ deficiency leads to G4 accumulation, DNA damage at G4-associated replication forks, and silencing of the RPA-mediated replication stress response. Our study provides first-hand evidence of the intrinsic, RF-coupled formation of G4 structures, offering unique mechanistic insights into the interference and regulation of stable G4s at replication forks and their effect on RPA-associated fork signaling and genomic instability.
PMID: 33953191
ISSN: 2041-1723
CID: 4866522