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 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 the mitosis-enabled anchor-away/recruiter system 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.

Profiling Tel1 Signaling Reveals a Non-Canonical Motif Targeting DNA Repair and Telomere Control Machineries.

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.

In-depth mapping of DNA-PKcs signaling uncovers noncanonical features of its kinase specificity.

DNA-PKcs is a DNA damage sensor kinase with established roles in DNA double-strand break repair via nonhomologous 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 noncanonical mode of DNA-PKcs signaling targeting S/T-ψ-D/E motifs. Sequence and structural analyses of the DNA-PKcs substrate recognition pocket revealed unique features compared to closely related phosphatidylinositol 3-kinase-related kinases that may explain its broader substrate preference. These findings expand the repertoire of DNA-PKcs and ATM substrates while establishing a novel preferential phosphorylation motif for DNA-PKcs.

Multi-step control of homologous recombination via Mec1/ATR suppresses 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 and phosphorylated by Mec1 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.

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.

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.

The DNA helicase FANCJ (BRIP1) functions in double strand break repair processing, but not crossover formation during prophase I of meiosis in male mice.

Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs result in crossovers (COs), sites of physical DNA exchange between homologs that are critical to correct chromosome segregation. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers, the latter representing the defining marks of CO sites. The regulation of CO number and position is poorly understood, but undoubtedly requires the coordinated action of multiple repair pathways. In a previous report, we found gene-trap disruption of the DNA helicase, FANCJ (BRIP1/BACH1), elicited elevated numbers of MLH1 foci and chiasmata. In somatic cells, FANCJ interacts with numerous DNA repair proteins including MLH1, and we hypothesized that FANCJ functions with MLH1 to regulate the major CO pathway. To further elucidate the meiotic function of FANCJ, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, truncation of the N-terminal Helicase domain, and a C-terminal dual-tagged allele. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, none of our Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 in meiosis. Instead, FANCJ co-localizes with BRCA1 and TOPBP1, forming discrete foci along the chromosome cores beginning in early meiotic prophase I and densely localized to unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data indicate a role for FANCJ in early DSB repair, but they rule out a role for FANCJ in MLH1-mediated CO events.

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-ψ-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.

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.

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.

ATR takes a crack at the nuclear envelope.

In this issue, Joo et al.1 and Kovacs et al.2 report that the ATR kinase promotes nuclear envelope rupture through the phosphorylation of Lamin A/C, inducing processes such as cGAS-STING pathway activation, micronuclei clearance, and potentially cell death.

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 the mediator proteins Rad9 and Mrc1. We demonstrate that Rad53 is phosphorylated and active following MMS exposure in cells lacking Mec1 and Tel1. This noncanonical 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.

DNA-PKcs promotes fork reversal and chemoresistance.

The DNA-PKcs kinase mediates the repair of DNA double-strand breaks via classical non-homologous end joining (NHEJ). DNA-PKcs is also recruited to active replication forks, although a role for DNA-PKcs in the control of fork dynamics is unclear. Here, we identify a crucial role for DNA-PKcs in promoting fork reversal, a process that stabilizes stressed replication forks and protects genome integrity. DNA-PKcs promotes fork reversal and slowing in response to several replication stress-inducing agents in a manner independent of its role in NHEJ. Cells lacking DNA-PKcs activity show increased DNA damage during S-phase and cellular sensitivity to replication stress. Notably, prevention of fork slowing and reversal via DNA-PKcs inhibition efficiently restores chemotherapy sensitivity in BRCA2-deficient mammary tumors with acquired PARPi resistance. Together, our data uncover a new key regulator of fork reversal and show how DNA-PKcs signaling can be manipulated to alter fork dynamics and drug resistance in cancer.

Proximity Labeling Reveals Spatial Regulation of the Anaphase-Promoting Complex/Cyclosome by a Microtubule Adaptor.

The anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination events. 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. We describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. Microtubule-localized proximity biotinylation tools revealed that PLEKHA5 depletion decreased APC/C association with the microtubule cytoskeleton, which prevented efficient loading of APC/C with its coactivator CDC20 and led to reduced APC/C E3 ligase activity. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5-APC/C interaction in microtubules. 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.

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 frequently exhibit Akt-driven 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 reflected gene expression changes characteristic of human breast tumors with higher LMNA expression, and specifically affected pathways related to cell-ECM interactions, cell metabolism, and PI3K/Akt signaling. 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, Akt signaling, and decreased disease-free survival. These findings suggest that downregulation of lamin A/C in breast cancer cells may influence both cellular physical properties and biochemical signaling to promote metastatic progression.

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis.

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

Phosphoproteomics of ATR signaling in mouse testes.

The phosphatidylinositol 3' kinase (PI3K)-related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression.

Fe-NTA Microcolumn Purification of Phosphopeptides from Immunoprecipitation (IP) Eluates for Mass Spectrometry Analysis.

Protein phosphorylation is a nearly universal signaling mechanism. To date, a number of proteomics tools have been developed to analyze phosphorylation. Phosphoproteome-wide analyses using whole cell extracts suffer from incomplete coverage, often missing phosphorylation events from low-abundance proteins. In order to increase coverage of phosphorylation sites on individual proteins of interest ("phospho-mapping"), immunoprecipitation (IP) followed by phosphoenrichment is necessary. Unfortunately, most commercially available phosphoenrichment kits are not readily scalable to the low-microgram quantities of protein present in IP eluates. Here, we describe a simple method specifically optimized for the enrichment of phosphopeptides from IP samples using an Fe-NTA based method. This method can be added downstream of any standard immunoprecipitation protocol and upstream of any MS analysis pipeline. The protocol described herein is cost effective, uses commonly available laboratory reagents, and can be used to obtain deep coverage of individual protein phosphorylation patterns, supplementary to phosphoproteomics data. Graphical abstract: Phospho-mapping workflow for a hypothetical protein of interest.

Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.

Structural basis of TRAPPIII-mediated Rab1 activation.

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo-EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α-helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.