Biology subjects

Sanford, E. J.

Publications and source records attributed to Sanford, E. J..

11 recordsLinked to original sources

bioRxiv2025

Enhanced protein precipitation with ammonia enables rapid, universal extraction of oligonucleotides for bioanalysis

Extracting oligonucleotides from biological matrices for mass spectrometry (MS) using current methodologies is time-consuming and costly. Accelerating the discovery, characterization, and development of oligonucleotides (ONTs) requires a universal method that is low-cost, efficient, without requiring extensive method development for each new test article. Protein precipitation using organic solvents meets these criteria for small molecules but has historically failed for ONTs due to poor recovery arising from their tendency to co-precipitate with proteins. This study introduces an effective approach utilizing small amines dissolved in organic solvents to significantly boost extraction recovery of ONTs from biological matrices .We first present the methods development and then analytically qualify it for the bioanalysis of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) extracted from various tissues, using ion-pairing reverse phase (IPRP) liquid chromatography coupled to tandem MS and high resolution MS (HRMS) detection. This method, Enhanced Protein Precipitation (EPP), achieves near-complete recovery across multiple ONT classes from biological matrices without need of sample digestion, costly solid phase extraction plates, or custom-designed hybridization probes. The method has proven to be more versatile and sustainable than conventional approaches. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/650521v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@a623d2org.highwire.dtl.DTLVardef@179b053org.highwire.dtl.DTLVardef@27273corg.highwire.dtl.DTLVardef@80fe47_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

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↗

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↗

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

Fork Slowing and Reversal as an Adaptive Response to Chronic ATR Inhibition

Inhibitors of the replication stress response kinase ATR are currently being explored in anti-cancer therapy. Acute ATR inhibition is known to impair the proper control of origin firing, DNA repair, and cell cycle, resulting in DNA breaks and mitotic catastrophe. Less is understood about the effects of clinically relevant regimes of ATR inhibition, which involve chronic and low doses of ATR inhibitors (cATRi) to cells. Here we report unexpected molecular effects of cATRi on replication dynamics. cATRi strongly reduces fork speed but has minimal effects on the accumulation of DNA breaks or cell survival. cATRi promotes extensive fork reversal and RAD51- and PARP-mediated fork slowing that correlate with the accumulation of DNA-RNA hybrids. Our work shows that fork reversal is a critical adaptive response ensuring cell survival during cATRi and that the manipulation of fork reversal causes hypersensitivity to cATRi, increasing the effectiveness of ATR inhibitors in anti-cancer therapies.

cancer biology↗

bioRxiv2020

Structure and mechanism of TRAPPIII-mediated Rab1 activation

The GTPase Rab1 is a master regulator of both the early secretory pathway and autophagy. Rab1 activation is controlled by its GEF (guanine nucleotide exchange factor), the multi-subunit TRAPPIII complex. The Trs85 regulatory subunit is critical for robust activation of Rab1 but its mechanistic role within the complex has remained unclear. Here we report the cryo-EM structure of the intact yeast TRAPPIII complex bound to its substrate Rab1/Ypt1. The orientation of the Rab1/Ypt1 hypervariable domain when bound to the complex leads to a model for how TRAPPIII associates with and activates Rab1/Ypt1 at the membrane surface. We identify a conserved amphipathic -helix motif within Trs85 and demonstrate that this helix is required for stable membrane binding and Rab1/Ypt1 activation by TRAPPIII. Taken together, our results provide a comprehensive analysis of the structure and function of the yeast TRAPPIII complex and reveal that the key function of Trs85 is to serve as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex.

cell biology↗

bioRxiv2020

Phosphoproteomics Reveals a Distinct Mode of Mec1/ATR Signaling in Response to DNA End Hyper-Resection

The Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, although how it promotes context-dependent signaling and DNA repair remains elusive. Here we uncovered a specialized mode of Mec1/ATR signaling triggered by the extensive nucleolytic processing (resection) of DNA ends. Cells lacking RAD9, a checkpoint activator and an inhibitor of resection, exhibit a selective increase in Mec1-dependent phosphorylation of proteins associated with single strand DNA transactions, including the ssDNA binding protein Rfa2, the translocase/ubiquitin ligase Uls1 and the HR-regulatory Sgs1-Top3-Rmi1 (STR) complex. Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper-resection to influence recombination outcomes. Overall, the identification of a distinct mode of Mec1 signaling triggered by hyper-resection highlights the multi-faceted action of this kinase in the coordination of checkpoint signaling and HR-mediated DNA repair.

molecular biology↗

bioRxiv2020

Maximizing Quantitative Phosphoproteomics of Kinase Signaling Expands the Mec1 and Tel1 Networks

Global phosphoproteome analysis is crucial for comprehensive and unbiased investigation of kinase-mediated signaling. However, since each phosphopeptide represents a unique entity for defining identity, site-localization, and quantitative changes, phosphoproteomics often suffers from lack of redundancy and statistical power for generating high confidence datasets. Here we developed a phosphoproteomic approach in which data consistency among experiments using reciprocal stable isotope labeling defines a central filtering rule for achieving reliability in phosphopeptide identification and quantitation. We find that most experimental error or biological variation in phosphopeptide quantitation does not revert in quantitation once light and heavy media are swapped between two experimental conditions. Exclusion of non-reverting data-points from the dataset not only reduces quantitation error and variation, but also drastically reduces false positive identifications. Application of our approach in combination with extensive fractionation of phosphopeptides by HILIC identifies new substrates of the Mec1 and Tel1 kinases, expanding our understanding of the DNA damage signaling network regulated by these kinases. Overall, the proposed quantitative phosphoproteomic approach should be generally applicable for investigating kinase signaling networks with high confidence and depth.

molecular biology↗

bioRxiv2019

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, Calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analysis demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

biochemistry↗

bioRxiv2019

The yeast Art1 arrestin domain contains disordered insertions that regulate its localization and activity

The protein composition of the plasma membrane is rapidly remodeled in response to changes in nutrient availability or cellular stress. This occurs, in part, through the selective ubiquitylation and endocytosis of plasma membrane proteins which, in the yeast Saccharomyces cerevisiae, is mediated by the HECT E3 ubiquitin ligase Rsp5 and arrestin-related trafficking (ART) adaptors. Here, we provide evidence that an ART family member, Art1, consists of an arrestin fold with extended N- and C-terminal tails, and interspersed with loop insertions. These loop and tail regions, while not strictly required for Art1 function, regulate its activity through two separate mechanisms. One loop mediates Art1 cargo specificity. Other loops are subjected to phosphorylation in a manner dependent on the Pho85 cyclins Clg1 and Pho80. Phosphorylation of the loops controls Art1s localization to the plasma membrane, which promotes cargo ubiquitylation and endocytosis, demonstrating a mechanism through which Art1 activity is regulated.

cell biology↗