Biology subjects

Rothenberg, E.

Publications and source records attributed to Rothenberg, E..

12 recordsLinked to original sources

bioRxiv2026

SHP2 binds directly to SOS1 to enable RAS activation

The protein-tyrosine phosphatase SHP2 (PTPN11) regulates growth factor- and cytokine-induced RAS/ERK MAP kinase (MAPK) pathway activation, and aberrant SHP2 function causes developmental disorders and cancer1-5. It is widely believed that the catalytic activity of SHP2 is essential for pathway activation1,4,6-8. This view has shaped our interpretation of how germline PTPN11 mutations cause Noonan Syndrome (NS) and NS with Multiple Lentigines (NS-ML)2,9 and how somatic mutations contribute to myeloproliferative neoplasms and solid tumors1. Here we identify a previously undetected, protein-tyrosine phosphatase (PTP) activity-independent mechanism that revises our understanding of how SHP2 promotes RAS/ERK activation. We find that certain mutations of the nucleophilic cysteine that abolish catalytic activity still promote RAS/ERK pathway activation in normal and neoplastic mammalian cells, zebrafish embryos, and mice. Structural studies show that the SHP2 PTP domain binds directly to the Son of Sevenless 1 (SOS1) Dbl homology (DH) domain. Proximity labeling and super-resolution microscopy demonstrate that SHP2/SOS1 interaction occurs in cells and facilitates SOS1 translocation to the plasma membrane to form clusters. Our results overturn decades of dogma on SHP2 regulation of the RAS/ERK pathway and provide new insights into the mechanism of action of disease-associated PTPN11 mutations.

Cancer Biology↗

bioRxiv2025

Hierarchical Coordination of Polymerase Theta and RAD51 Resolves Clustered Replication Fork Collapse

The essential role of polymerase theta (Pol{theta})-mediated end joining (TMEJ), an alternative double strand break repair pathway, has been primarily studied in homologous recombination (HR)-deficient contexts(1, 2). Here, we uncover an indispensable role for TMEJ in HR-proficient mammalian cells during the repair of interstrand crosslinks (ICLs). We show that Pol{theta} is recruited downstream of canonical ICL repair steps--including ICL unhooking, RAD51 loading, and RAD51 ubiquitylation--and localizes to sites of unresolved HR through interactions with ubiquitylated RAD51 filaments. Using genomic scar profiling and targeted ICL repair assays, we find that TMEJ resolves a minor subset of lesions that are not amenable to HR repair, such as clustered ICLs that can induce two-ended replication fork collapse. These findings reveal a RAD51 ubiquitylation-dependent mechanism for Pol{theta} recruitment and establish TMEJ as a hierarchically deployed DNA repair pathway that safeguards genome stability when HR is insufficient to resolve replication-associated DNA damage. Short SummaryPol{theta} is recruited via RAD51 ubiquitylation to resolve clustered ICLs that generate HR-refractory replication fork collapse.

cell biology↗

bioRxiv2024

The KU70-SAP domain has an overlapping function with DNA-PKcs in limiting the lateral movement of KU along DNA

The non-homologous end-joining (NHEJ) pathway is critical for DNA double-strand break repair and is essential for lymphocyte development and maturation. The Ku70/Ku80 heterodimer (KU) binds to DNA ends, initiating NHEJ and recruiting additional factors, including DNA-dependent protein kinase catalytic subunit (DNA-PKcs) that caps the ends and pushes KU inward. The C-terminus of Ku70 in higher eukaryotes includes a flexible linker and a SAP domain, whose physiological role remains poorly understood. To investigate this, we generated a mouse model with knock-in deletion of the SAP domain (Ku70{Delta}SAP/{Delta}SAP). Ku70{Delta}SAP supports KU stability and its recruitment to DNA damage sites in vivo. In contrast to the growth retardation and immunodeficiency seen in Ku70-/- mice, Ku70{Delta}SAP/{Delta}SAP mice show no defects in lymphocyte development and maturation. Structural modeling of KU on long dsDNA, but not dsRNA suggests that the SAP domain can bind to an adjacent major groove, where it can limit KUs rotation and lateral movement along the dsDNA. Accordingly, in the absence of DNA-PKcs that caps the ends, Ku70{Delta}SAP fails to support stable DNA damage-induced KU foci. In DNA-PKcs-/- mice, Ku70{Delta}SAP abrogates the leaky T cell development and reduces both the qualitative and quantitative aspects of residual V(D)J recombination. In the absence of DNA-PKcs, purified Ku70{Delta}SAP has reduced affinity for DNA ends and dissociates more readily at lower concentration and accumulated as multimers at high concentration. These findings revealed a physiological role of the SAP domain in NHEJ by restricting KU rotation and lateral movement on DNA that is largely masked by DNA-PKcs. HighlightKu70 is a conserved non-homologous end-joining (NHEJ) factor. Using genetically engineered mouse models and biochemical analyses, our study uncovered a previously unappreciated role of the C-terminal SAP domain of Ku70 in limiting the lateral movement of KU on DNA ends and ensuring end protection. The presence of DNA-PKcs partially masks this role of the SAP domain.

genetics↗

bioRxiv2024

Inactive Parp2 causes Tp53-dependent lethal anemia by blocking replication-associated nick ligation in erythroblasts

PARP1&2 enzymatic inhibitors (PARPi) are promising cancer treatments. But recently, their use has been hindered by unexplained severe anemia and treatment-related leukemia. In addition to enzymatic inhibition, PARPi also trap PARP1&2 at DNA lesions. Here, we report that unlike Parp2-/- mice, which develop normally, mice expressing catalytically-inactive Parp2 (E534A, Parp2EA/EA) succumb to Tp53- and Chk2-dependent erythropoietic failure in utero, mirroring Lig1-/- mice. While DNA damage mainly activates PARP1, we demonstrate that DNA replication activates PARP2 robustly. PARP2 is selectively recruited and activated by 5-phosphorylated nicks (5p-nicks) between Okazaki fragments, typically resolved by Lig1. Inactive PARP2, but not its active form or absence, impedes Lig1- and Lig3-mediated ligation, causing dose-dependent replication fork collapse, particularly harmful to erythroblasts with ultra-fast forks. This PARylation-dependent structural function of PARP2 at 5p-nicks explains the detrimental effects of PARP2 inhibition on erythropoiesis, revealing the mechanism behind the PARPi-induced anemia and leukemia, especially those with TP53/CHK2 loss. SignificanceThis work shows that the hematological toxicities associated with PARP inhibitors stem not from impaired PARP1 or PARP2 enzymatic activity but rather from the presence of inactive PARP2 protein. Mechanistically, these toxicities reflect a unique role of PARP2 at 5-phosphorylated DNA nicks during DNA replication in erythroblasts.

genetics↗

bioRxiv2023

ORC1 binds to cis-transcribed RNAs for efficient activation of replication origins

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. We propose that fluctuating concentrations of RNA during the cell cycle may play a sequential role in controlling origins through interaction with this flexible region of ORC1. Our results unveil a novel non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins. One sentence summaryThe human origin recognition complex subunit 1 ORC1, binds to RNAs transcribed from genes with origins of replication at the TSS, which is required for optimal origin activation.

molecular biology↗

bioRxiv2022

The non-catalytic role of DNA polymerase epsilon in replication initiation in human cells

DNA polymerase epsilon in an essential enzyme, responsible for the synthesis of the leading strand during DNA replication. Deficiencies and mutations in DNA polymerase epsilon catalytic subunit (POLE1) cause severe developmental abnormalities and cancers. Paradoxically, the non-catalytic C-terminal domain of yeast polymerase epsilon catalytic subunit (Pol2) is sufficient for cell survival. The non-catalytic essential function of Pol2 in yeast has been associated with its role in the assembly of the replicative helicase CMG. However, the understanding of POLE1 functions in DNA replication initiation in human cells is falling behind. In this study we use an auxin-inducible degron system to study the effect of POLE1 depletion on replication initiation in human cells. Surprisingly, in the absence of POLE1, human cells were able to assemble CMG helicase and initiate DNA synthesis that failed shortly after. Expression of POLE1 C-terminal non-catalytic domain was enough to rescue replication initiation and support slow, but processive DNA synthesis, which was dependent on the POLE1-POLE2 interaction. We propose a model where in human cells POLE1/POLE2 are not essential for CMG assembly, but are required during later steps of replication initiation. Our study provides some insights into the role of DNA polymerase epsilon in replication initiation in human cells.

cell biology↗

bioRxiv2020

Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

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-treatment in human cancer cell-line 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 more active/functional segments. Data were corroborated by colocalization studies via dSTORM microscopy. This approach can also be applied to study the dynamic spatio-temporal nature of genomic uracil.

biochemistry↗

bioRxiv2020

Super-resolution visualization of distinct stalled and broken replication fork structures

Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork (RF) stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). To avoid mutagenesis, these breaks require repair via Homologous Recombination (HR). Here we apply multicolor single molecule super resolution microscopy to visualize individual RFs under mild stress from the trapping of Topoisomerase I cleavage complexes, a damage induction which closely mimics endogenous replicative stress. We identify RAD51 and RAD52, alongside RECQ1, as the first responder proteins to stalled but unbroken forks, whereas Ku and MRE11 are initially recruited to seDSBs. Ku loads directly onto the DSB end whereas MRE11 associates with nascent DNA away from the break, and both proteins colocalize contemporaneously with a single seDSB. We are thus able to discern closely related RF stress motifs and their repair pathways in vivo, uncovering mechanistic insights into the nature of RF damage and repair.

genomics↗

bioRxiv2019

PARPi synthetic lethality derives from replication-associated single-stranded DNA gaps

BRCA1 or BRCA2 (BRCA)-deficient tumor cells have defects in DNA double strand break repair by homologous recombination (HR) and fork protection (FP) that are thought to underlie the sensitivity to poly(ADP-ribose) polymerase inhibitor (PARPi). Given the recent finding that PARPi accelerates DNA replication, it was proposed that high speed DNA replication leads to DNA double strand breaks (DSBs). Here, we tested the alternative hypothesis that PARPi sensitivity in BRCA deficient cells results from combined replication dysfunction that causes a lethal accumulation of replication-associated single-stranded DNA (ssDNA) gaps. In support of a gap toxicity threshold, PARPi-induced ssDNA gaps accumulate more excessively in BRCA deficient cells and are suppressed in de novo and genetic models of PARPi resistance while defects in HR or FP often lack this correlation. We also uncouple replication speed from lethality. The clear link between PARPi sensitivity and ssDNA gaps provides a new paradigm for understanding synthetic lethal interactions.

cancer biology↗

bioRxiv2019

Single-molecule localization of Nav1.5 reveals different modes of reorganization at the lateral membrane and T-tubules of cardiomyocytes

Mutations in the gene encoding the sodium channel Nav1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Nav1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Nav1.5 localization and function remain insufficiently characterized. We used novel single-molecule localization microscopy (SMLM) and modeling to define nanoscale features of Nav1.5 localization and distribution at the lateral membrane, groove, and T-tubules in wild-type, dystrophin-deficient (mdx) mice, and mice expressing C-terminally truncated Nav1.5 ({Delta}SIV). We show that Nav1.5 organizes as distinct clusters in the groove and T-tubules which density and distribution partially depend on SIV and dystrophin. We found that overall reduction in Nav1.5 expression in mdx and {Delta}SIV cells results in a non-uniform redistribution with Nav1.5 being specifically reduced at the groove of {Delta}SIV and increased in T-tubules of mdx cardiomyocytes. Nav1.5 mutations may therefore site-specifically affect Nav1.5 localization and distribution depending on site-specific interacting proteins.

physiology↗

bioRxiv2018

A role for RNA and DNA:RNA hybrids in the modulation of DNA repair by homologous recombination

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 and promote its recruitment to DSBs. We also show that RNase H2 is in a complex with the HR proteins BRCA1, PALB2, BRCA2, and RAD51, and that it localizes to DSBs in the S/G2 cell-cycle phase. BRCA2 controls DNA:RNA hybrid levels at DSBs by mediating RNase H2 recruitment and, therefore, hybrids degradation. These results demonstrate that regulated DNA:RNA hybrid levels at DSBs contribute to HR-mediated repair.

cell biology↗

bioRxiv2017

A Method for Quantifying Molecular Interactions Using Stochastic Modelling and Super-Resolution Microscopy

We introduce the Interaction Factor (IF), a measure for quantifying the interaction of molecular clusters in super-resolution microscopy images. The IF is robust in the sense that it is independent of cluster density, and it only depends on the extent of the pair-wise interaction between different types of molecular clusters in the image. The IF for a single or a collection of images is estimated by first using stochastic modelling where the locations of clusters in the images are repeatedly randomized to estimate the distribution of the overlaps between the clusters in the absence of interaction (IF=0). Second, an analytical form of the relationship between IF and the overlap (which has the random overlap as its only parameter) is used to estimate the IF for the experimentally observed overlap. The advantage of IF compared to conventional methods to quantify interaction in microscopy images is that it is insensitive to changing cluster density and is an absolute measure of interaction, making the interpretation of experiments easier. We validate the IF method by using both simulated and experimental data and provide an ImageJ plugin for determining the IF of an image.

bioinformatics↗