Biology subjects

Smolka, M. B.

Publications and source records attributed to Smolka, M. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Kcs1-dependent inositol pyrophosphates promote DNA double-strand break repair via Pho80-Pho85 in budding yeast

The cleavage of membrane-bound phosphoinositides produces inositol polyphosphates (IPs) and pyrophosphates (PP-IPs), small water-soluble signaling molecules that are highly conserved in eukaryotes and play a critical role in cell metabolism. Loss of the inositol pyrophosphate kinase (PP-IPk) Kcs1 and its mammalian ortholog IP6K1 was previously shown to cause hypersensitivity to genotoxic stress and reduced homologous recombination (HR) repair. To identify the mechanism linking PP-IPs to DNA double-strand break (DSB) repair, we combined quantitative proteomics with biochemical, genetic, and cell-cycle analyses in budding yeast. We found that loss of Kcs1, but not the PP-IPk Vip1, caused increased sensitivity to the radiomimetic drug zeocin. kcs1{Delta} cells displayed persistent Rad53 signaling and elevated {gamma}-H2A levels, remaining arrested in G1/S during treatment and accumulating in G2/M for hours after damage removal. Unexpectedly, Kcs1-dependent PP-IPs promoted DSB repair independently of the core HR machinery and of cell-cycle stage. Quantitative proteomics revealed increased levels of PHO pathway proteins in zeocin treated kcs{Delta} cells, and deletion of the CDK inhibitor PHO81 substantially rescued zeocin hypersensitivity, indicating that constitutive inhibition of the Pho80-Pho85 cyclin-dependent kinase underlies the DNA repair defect caused by Kcs1 loss. This function of Pho80-Pho85 was mechanistically distinct from its canonical role in phosphate homeostasis, and partially independent of its connections to Ku80 regulation. Together, our findings identify Kcs1-dependent PP-IP signaling as a new upstream input controlling Pho80-Pho85 activity during the DNA damage response, establishing an HR-independent axis for genome maintenance and linking PP-IP metabolism to the DNA damage response in eukaryotic cells.

cell biology↗

bioRxiv2026

Non-canonical ATR signalling via NBS1 phosphorylation propagates fork slowing from stressed to unperturbed nuclear regions

DNA replication forks frequently encounter obstacles and remodel into four-way junctions to actively slow DNA fork progression. Fork slowing can also spread to undamaged forks via an ATR-dependent mechanism that remained elusive. Here, using mild genotoxic stress, we show that fork slowing and reversal require full ATR activity, but no canonical ATR activators and signalling partners, defining a non-canonical ATR pathway distinct from origin firing control. Phospho-proteomics in S-phase cells under checkpoint-blind replication stress identified a subset of ATR-dependent phospho-sites, such as S343 on NBS1, the regulatory subunit of the MRN complex. This residue is essential to stimulate MRN exonuclease activity in vitro and required in cells for global fork slowing upon mild DNA damage. Ultimately, local UV-C micro-irradiation reveals that ATR-dependent MRN-stimulated resection dampens DNA synthesis at lesions and propagates fork slowing to undamaged chromatin, supporting MRN-mediated ssDNA exposure as a mean to coordinate replication slowdown across the nucleus.

cell biology↗

bioRxiv2026

Digital Chromosome Banding Reveals Distinct Spatiotemporal Dynamics and Sexual Dimorphism in Meiotic Silencing

In mammals, meiotic silencing of unsynapsed chromatin (MSUC) is initiated by the DNA damage response (DDR) pathway, as marked by {gamma}H2AX. During normal male meiosis, MSUC is restricted to the unsynapsed sex chromosomes, a process known as meiotic sex chromosome inactivation (MSCI). While the initiation of MSCI has been well studied, its full silencing dynamics and underlying structural mechanisms remain unclear. In contrast to MSCI, broader MSUC can occur on autosomes in response to synapsis failure, but its cell-to-cell variability obscures its quantification. To address these challenges, we introduce "digital-chromosome-banding", a single-cell-based approach that allows quantitative analysis of MSCI and MSUC at chromosomal resolution. Using this approach, we identified two distinct silencing transitions during MSCI, occurring from zygonema to early pachynema and from early to mid-pachynema. The latter step coincides with mature sex body formation and involves a gel-like diffusion barrier to enforce transcriptional repression. Applying this approach to synapsis-defective mouse models (Spo11-/-, Tardbp cKO, and Nelfb cKO), we observed divergent MSUC patterns that correlate with the severity of asynapsis. Comparative analysis between sexes also uncovered notable sexual dimorphisms in meiotic silencing. Together, our data provide a quantitative framework to dissect the spatiotemporal dynamics and sexual differences of meiotic silencing.

cell biology↗

bioRxiv2025

An ATM-PPM1D Circuit Controls the Processing and Restart of DNA Replication Forks

In response to DNA replication stress, DNA damage signaling kinases inhibit origin firing and promote the remodeling and stabilization of replication forks, leading to a systemic reduction in DNA synthesis, which collectively protects genomic integrity. Little is understood about the regulatory mechanisms of replication stress recovery, including the mechanisms involved in the restart of stalled replication forks. Here, we identify the oncogenic phosphatase PPM1D/WIP1 as a regulator of replication fork restart. During recovery from replication stress, PPM1D prevents excessive MRE11- and DNA2-dependent nucleolytic degradation of stalled forks. Loss of PPM1D function leads to defects in RAD51 recruitment to chromatin and impairs RAD51-dependent fork restart. Phosphoproteomic analysis reveals that PPM1D regulates a network of ATM substrates, several of which are phosphorylated at an S/T-Q-(E/D)n motif. Strikingly, inhibition of ATM suppresses the deleterious consequences of impaired PPM1D function at replication forks, enabling timely fork restart. The dominant effect of ATM hyper-signaling in suppressing fork restart occurs, in part, through the excessive engagement of 53BP1 and consequent RAD51 antagonization. These findings uncover a new mode of ATM signaling responding to fork stalling and highlight the need for PPM1D to restrain ATM signaling and facilitate proper fork restart.

biochemistry↗

bioRxiv2024

Proteomic Sensors for Quantitative, Multiplexed and Spatial Monitoring of Kinase Signaling

Understanding kinase action requires precise quantitative measurements of their activity in vivo. In addition, the ability to capture spatial information of kinase activity is crucial to deconvolute complex signaling networks, interrogate multifaceted kinase actions, and assess drug effects or genetic perturbations. Here we developed a proteomic kinase activity sensor platform (ProKAS) for the analysis of kinase signaling using mass spectrometry. ProKAS is based on a tandem array of peptide sensors with amino acid barcodes that allow multiplexed analysis for spatial, kinetic, and screening applications. We engineered a ProKAS module to simultaneously monitor the activities of the DNA damage response kinases ATR, ATM, and CHK1 in response to genotoxic drugs, while also uncovering differences between these signaling responses in the nucleus, cytosol, and replication factories. Furthermore, we developed an in silico approach for the rational design of specific substrate peptides expandable to other kinases. Overall, ProKAS is a novel versatile system for systematically and spatially probing kinase action in cells.

molecular biology↗

bioRxiv2024

High-Resolution Genome-Wide Mapping of Chromatin Accessibility and Torsional Stress

Torsional stress in chromatin plays a fundamental role in cellular functions, influencing key processes such as transcription, replication, and chromatin organization. Transcription and other processes may generate and be regulated by torsional stress. In the genome, the interplay of these processes creates complicated patterns of both positive (+) and negative (-) torsion. However, a challenge in generating an accurate torsion map is determining the zero-torsion baseline signal, which is conflated with chromatin accessibility. Here, we introduce a high-resolution method based on the intercalator trimethylpsoralen (TMP) to address this challenge. We describe a method to establish the zero-torsion baseline while preserving the chromatin state of the genome of S. cerevisiae. This approach enables both high-resolution mapping of accessibility and torsional stress in chromatin in the cell. Our analysis shows transcription-generated torsional domains consistent with the twin-supercoiled-domain model of transcription and suggests a role for torsional stress in recruiting topoisomerases and in regulating 3D genome architecture via cohesin. Significantly, we reveal that insulator sequence-specific transcription factors decouple torsion between divergent promoters, whereas torsion spreads between divergent promoters lacking these factors, suggesting that torsion serves as a regulatory mechanism in these regions. Although insulators are known to decouple gene expression, our finding provides a physical explanation of how such decoupling may occur. This new method provides a potential path forward for using TMP to measure torsional stress in the genome without the confounding contribution of accessibility in chromatin.

genomics↗

bioRxiv2024

High Coverage Profiling of Tel1 Signaling Reveals a Predominant Non-Canonical Phospho-Motif

The stability of the genome relies on Phosphatidyl Inositol 3-Kinase-related Kinases (PIKKs) that sense DNA damage and trigger elaborate downstream signaling responses. In S. cerevisiae, the Tel1 kinase (ortholog of human ATM) is activated at DNA double strand breaks (DSBs) and short telomeres. Despite the well-established roles of Tel1 in the control of telomere maintenance, suppression of chromosomal rearrangements, activation of cell cycle checkpoints, and repair of DSBs, the substrates through which Tel1 controls these processes remain incompletely understood. Here we performed an in depth phosphoproteomic screen for Tel1-dependent phosphorylation events. To achieve maximal coverage of the phosphoproteome, we developed a scaled-up approach that accommodates large amounts of protein extracts and chromatographic fractions. Compared to previous reports, we expanded the number of detected Tel1-dependent phosphorylation events by over 10-fold. Surprisingly, in addition to the identification of phosphorylation sites featuring the canonical motif for Tel1 phosphorylation (S/T-Q), the results revealed a novel motif (D/E-S/T) highly prevalent and enriched in the set of Tel1-dependent events. This motif is unique to Tel1 signaling and not shared with the Mec1 kinase, providing clues to how Tel1 plays specialized roles in DNA repair and telomere length control. Overall, these findings define a Tel1-signaling network targeting numerous proteins involved in DNA repair, chromatin regulation, and telomere maintenance that represents a framework for dissecting the molecular mechanisms of Tel1 action.

molecular biology↗

bioRxiv2024

MAGMa: Your Comprehensive Tool for Differential Expression Analysis in Mass-Spectrometry Proteomic Data.

Proteomics, the study of proteins and their functions, plays a vital role in understanding biological processes. In this study, we sought to address the challenges in analyzing complex proteomic datasets, where subtle changes in protein abundance are difficult to detect. Utilizing a newly developed tool, Maximal Aggregation of Good protein signal from Mass spectrometric data (MAGMa), we demonstrated its superior performance in accurately identifying true signals while effectively filtering out noise. Here we show that MAGMa strikes a balance between sensitivity and specificity on benchmarking datasets, offering a robust solution for analyzing various quantitative proteomic datasets. These findings advance the field by providing researchers with a powerful tool to uncover subtle changes in protein abundance, contributing to our understanding of complex biological systems and potentially facilitating the discovery of new therapeutic targets.

bioinformatics↗

bioRxiv2024

A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization

Tools for acute manipulation of protein localization enable elucidation of spatiotemporally defined functions, but their reliance on exogenous triggers can interfere with cell physiology. This limitation is particularly apparent for studying mitosis, whose highly choreographed events are sensitive to perturbations. Here we exploit the serendipitous discovery of a phosphorylation-controlled, cell cycle-dependent localization change of the adaptor protein PLEKHA5 to develop a system for mitosis-specific protein recruitment to the plasma membrane that requires no exogenous stimulus. Mitosis-enabled Anchor-away/Recruiter System (MARS) comprises an engineered, 15-kDa module derived from PLEKHA5 capable of recruiting functional protein cargoes to the plasma membrane during mitosis, either through direct fusion or via GFP-GFP nanobody interaction. Applications of MARS include both knock sideways to rapidly extract proteins from their native localizations during mitosis and conditional recruitment of lipid-metabolizing enzymes for mitosis-selective editing of plasma membrane lipid content, without the need for exogenous triggers or perturbative synchronization methods.

cell biology↗

bioRxiv2024

In-Depth Mapping of DNA-PKcs Signaling Uncovers Conserved Features of Its Kinase Specificity

DNA-PKcs is a DNA damage sensor kinase with established roles in DNA double-strand break repair via non-homologous end joining. Recent studies have revealed additional roles of DNA-PKcs in the regulation of transcription, translation and DNA replication. However, the substrates through which DNA-PKcs regulates these processes remain largely undefined. Here we utilized quantitative phosphoproteomics to generate a high coverage map of DNA-PKcs signaling in response to ionizing radiation and mapped its interplay with the ATM kinase. Beyond the detection of the canonical S/T-Q phosphorylation motif, we uncovered a non-canonical mode of DNA-PKcs signaling targeting S/T-{psi}-D/E motifs. Cross-species analysis in mouse pre-B and human HCT116 cell lines revealed splicing factors and transcriptional regulators phosphorylated at this novel motif, several of which contain SAP domains. These findings expand the list of DNA-PKcs and ATM substrates and establish a novel preferential phosphorylation motif for DNA-PKcs that connects it to proteins involved in nucleotide processes and interactions.

biochemistry↗

bioRxiv2023

Multi-Step Control of Homologous Recombination by Mec1/ATR Ensures Robust Suppression of Gross Chromosomal Rearrangements

The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.

genetics↗

bioRxiv2023

The DNA helicase FANCJ (BRIP1) functions in Double Strand Break repair processing, but not crossover formation during Prophase I of meiosis in male mice

During meiotic prophase I, recombination between homologous parental chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs), each of which must be repaired with absolute fidelity to ensure genome stability of the germline. One outcome of these DSB events is the formation of Crossovers (COs), the sites of physical DNA exchange between homologs that are critical to ensure the correct segregation of parental chromosomes. However, COs account for only a small ([~]10%) proportion of all DSB repair events; the remaining 90% are repaired as non-crossovers (NCOs), most by synthesis dependent strand annealing. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers. The number and positioning of COs is exquisitely controlled via mechanisms that remain poorly understood, but which undoubtedly require the coordinated action of multiple repair pathways downstream of the initiating DSB. In a previous report we found evidence suggesting that the DNA helicase and Fanconi Anemia repair protein, FANCJ (BRIP1/BACH1), functions to regulate meiotic recombination in mouse. A gene-trap disruption of Fancj showed an elevated number of MLH1 foci and COs. FANCJ is known to interact with numerous DNA repair proteins in somatic cell repair contexts, including MLH1, BLM, BRCA1, and TOPBP1, and we hypothesized that FANCJ regulates CO formation through a direct interaction with MLH1 to suppress the major CO pathway. To further elucidate the function of FANCJ in meiosis, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, a mutant line lacking the MLH1 interaction site and the N-terminal region of the Helicase domain, and a C-terminal 6xHIS-HA dual-tagged allele of Fancj. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, while Fanconi-like phenotypes are observed within the somatic cell lineages of the full deletion Fancj line, none of the Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I of meiosis. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 during late prophase I. Instead, FANCJ forms discrete foci along the chromosome cores beginning in early meiotic prophase I, occasionally co-localizing with MSH4, and then becomes densely localized on unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Strikingly, this localization strongly overlaps with BRCA1 and TOPBP1. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data suggest a role for FANCJ in early DSB repair events, and possibly in the formation of NCOs, but they rule out a role for FANCJ in MLH1-mediated CO events. Thus, the role of FANCJ in meiotic cells involves different pathways and different interactors to those described in somatic cell lineages.

genetics↗

bioRxiv2023

A TOPBP1 Allele Causing Male Infertility Uncouples XY Silencing Dynamics From Sex Body Formation

Meiotic sex chromosome inactivation (MSCI) is a critical feature of meiotic prophase I progression in males. While the ATR kinase and its activator TOPBP1 are key drivers of MSCI within the specialized sex body (SB) domain of the nucleus, how they promote silencing remains unclear given their multifaceted meiotic functions that also include DNA repair, chromosome synapsis and SB formation. Here we report a novel mutant mouse harboring mutations in the TOPBP1-BRCT5 domain. Topbp1B5/B5 males are infertile, with impaired MSCI despite displaying grossly normal events of early prophase I, including synapsis and SB formation. Specific ATR-dependent events are disrupted including phosphorylation and localization of the RNA:DNA helicase Senataxin. Topbp1B5/B5 spermatocytes initiate, but cannot maintain ongoing, MSCI. These findings reveal a non-canonical role for the ATR-TOPBP1 signaling axis in MSCI dynamics at advanced stages in pachynema and establish the first mouse mutant that separates ATR signaling and MSCI from SB formation.

cell biology↗

bioRxiv2023

Progranulin inhibits phospholipase sPLA2-IIA to control neuroinflammation

Withdrawal StatementThe authors have withdrawn this manuscript because more work is needed to fully define the role of sPLA2-IIA. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

neuroscience↗

bioRxiv2022

Mec1-Independent Activation of the Rad53 Checkpoint Kinase Revealed by Quantitative Analysis of Protein Localization Dynamics

The replication checkpoint is essential for accurate DNA replication and repair, and maintenance of genomic integrity when a cell is challenged with genotoxic stress. Several studies have defined the complement of proteins that change subcellular location in the budding yeast Saccharomyces cerevisiae following chemically-induced DNA replication stress using methyl methanesulfonate (MMS) or hydroxyurea (HU). How these protein movements are regulated remains largely unexplored. We find that the essential checkpoint kinases Mec1 and Rad53 are responsible for regulating the subcellular localization of 159 proteins during MMS-induced replication stress. Unexpectedly, Rad53 regulation of the localization of 52 proteins is independent of its known kinase activator, Mec1, and in some scenarios independent of Tel1, or mediator components, Rad9 and Mrc1. We demonstrate that Rad53 is phosphorylated and active following MMS exposure in cells lacking Mec1 and Tel1. This non-canonical mode of Rad53 activation depends partly on the retrograde signaling transcription factor Rtg3, which also facilitates proper DNA replication dynamics. We conclude that there are biologically important modes of Rad53 protein kinase activation that respond to replication stress and operate in parallel to Mec1 and Tel1.

genetics↗

bioRxiv2022

PLEKHA5 regulates mitotic progression by promoting APC/C localization to microtubules

The anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination of effector proteins. Despite the long-appreciated spatial organization of key events in mitosis mediated largely by cytoskeletal networks, the spatial regulation of APC/C, the major mitotic E3 ligase, is poorly understood. Here, we describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5-APC/C interaction. To assess the spatial importance of this interaction, we developed microtubule-localized proximity biotinylation tools, which revealed that depletion of PLEKHA5 decreased the extent of APC/C association with the microtubule cytoskeleton. This decreased APC/C microtubule-localization in turn prevented efficient loading of APC/C with its co-activator CDC20, leading to defects in E3 ligase catalytic activity. We propose that PLEKHA5 functions as an adaptor of APC/C that promotes its subcellular localization to microtubules and facilitates its activation by CDC20, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis.

cell biology↗

bioRxiv2021

Comprehensive analysis of DNA replication timing in genetic diseases and gene knockouts identifies MCM10 as a novel regulator of the replication program

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. MCM10 mutant cells demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in MCM10-mutant cells was predominantly comprised of replication initiation defects. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel modulator of DNA replication timing.

genetics↗

bioRxiv2021

Low lamin A levels enhance confined cell migration and metastatic capacity in breast cancer

Aberrations in nuclear size and shape are commonly used to identify cancerous tissue. However, it remains unclear whether the disturbed nuclear structure directly contributes to the cancer pathology or is merely a consequence of other events occurring during tumorigenesis. Here, we show that highly invasive and proliferative breast cancer cells have lower expression of the nuclear envelope proteins lamin A/C, leading to increased nuclear deformability that permits enhanced cell migration through confined environments that mimic interstitial spaces encountered during metastasis. Importantly, increasing lamin A/C expression in highly invasive breast cancer cells altered expression of numerous other proteins implicated in metastatic progression. Further supporting an important role of lamins in breast cancer metastasis, analysis of lamin levels in human breast tumors revealed a significant association between lower lamin A levels and decreased disease-free survival. These findings suggest that downregulation of lamin A/C may influence both biochemical and physical properties of the cell to promote breast cancer metastatic progression.

cancer biology↗