Biology subjects

Cobb, J. A.

Publications and source records attributed to Cobb, J. A..

8 recordsLinked to original sources

bioRxiv2025

Development of an inhibitory TTC7B selective nanobody that blocks EFR3 recruitment of PI4KA

Phosphatidylinositol 4 kinase III (PI4KIII/PI4KA) is an essential lipid kinase that plays a critical role in regulating plasma membrane identity. PI4KA is primarily recruited to the plasma membrane through the targeted recruitment by the proteins, EFR3A and EFR3B, which bind to the PI4KA accessory proteins TTC7 (TTC7A/B) and FAM126 (FAM126A/B). Here we characterised how both EFR3 isoforms interact with all possible TTC7-FAM126 combinations and developed a nanobody that specifically blocked EFR3-mediated PI4KA recruitment in TTC7B containing complexes. Most EFR3-TTC7-FAM126 combinations show similar binding affinities, with the exception of EFR3A-TTC7B-FAM126A, which binds with a [~]10-fold higher affinity. Moreover, we showed that EFR3B phosphorylation markedly decreased binding to TTC7-FAM126. Using a yeast display approach, we isolated a TTC7B selective nanobody that blocked EFR3 binding. Cryo-electron microscopy and hydrogen deuterium exchange mass spectrometry showed an extended interface with both PI4KA and TTC7B that sterically blocks EFR3 binding. The nanobody caused decreased membrane recruitment both on lipid bilayers and in cells, with decreased PM production of PI4P. Collectively, these findings provide new insights into PI4KA regulation and provide a tool for manipulating PI4KA complexes, that may be valuable for therapeutic targeting.

biochemistry↗

bioRxiv2023

Intrinsic disorder specifies discreet functions of nucleoplasmin-like histone chaperones

Nucleoplasmin (NPM) histone chaperones regulate distinct processes in the nucleus and nucleolus. While intrinsically disordered regions (IDRs) are hallmarks of NPMs, it is not clear if all NPM functions require these unstructured features. We assessed the importance of IDRs in yeast NPM-like proteins and found that regulation of rDNA copy number, and genetic interactions with the nucleolar RNA surveillance machinery require the highly conserved FKBP prolyl isomerase domain, but not the NPM domain or IDRs. By contrast, transcriptional silencing in the nucleus requires IDRs. We demonstrate that multiple lysines in poly-acidic serine/lysine motifs of IDRs are required for both lysine polyphosphorylation and NPM-mediated transcriptional silencing. These results demonstrate that NPM-like proteins do not rely on IDRs for all chromatin-related functions.

molecular biology↗

bioRxiv2022

SIR telomere silencing depends on nuclear envelope lipids and modulates sensitivity to a lysolipid drug

The nuclear envelope (NE) is important in maintaining genome organization. The role of lipids in the communication between the NE and telomere silencing was investigated, including how changes in lipid composition impact gene expression and overall nuclear architecture. For this purpose, yeast cells were treated with the non-metabolizable lysophosphatidylcholine analog edelfosine, known to accumulate at the perinuclear endoplasmic reticulum. Edelfosine treatment induced NE deformation and disrupted telomere clustering but not anchoring. In addition, the association of Sir4 at telomeres measured by ChIP decreased. RNA-seq analysis showed altered expression of Sir-dependent genes located at sub-telomeric (0-10 kb) regions, which was consistent with Sir4 dispersion. Transcriptomic analysis revealed that two lipid metabolic circuits were activated in response to edelfosine, one mediated by the membrane sensing transcription factors, Spt23/Mga2, and the other by a transcriptional repressor, Opi1. Activation of these combined transcriptional programs resulted in higher levels of unsaturated fatty acids and the formation of nuclear lipid droplets. Interestingly, cells lacking Sir proteins displayed resistance to unsaturated fatty acids and edelfosine, and this phenotype was connected to Rap1. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/499406v3_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@a46borg.highwire.dtl.DTLVardef@2a7f44org.highwire.dtl.DTLVardef@95ceforg.highwire.dtl.DTLVardef@b4678c_HPS_FORMAT_FIGEXP M_FIG C_FIG SummaryThe nuclear envelope (NE) is important for nuclear organization. This study shows that changes in NE lipid composition from lysolipid treatment decreases Sir4 association with telomeres, their clustering at NE, and triggers lipid-specific transcriptional circuits regulated by membrane-sensing factors.

molecular biology↗

bioRxiv2022

During replicative aging, changes in DNA double-strand break repair correlate with an increased rate of mutation.

A double -strand break (DSB) is one of the most deleterious forms of DNA damage. In eukaryotic cells, two main repair pathways have evolved to repair DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is the predominant pathway of repair in the unicellular eukaryotic organism, S. cerevisiae. However, during replicative aging the relative use of HR and NHEJ shifts in favor of end-joining repair. By monitoring repair events in the HO-DSB system, we find that early in replicative aging there is a decrease in the association of long-range resection factors, Dna2-Sgs1 and Exo1 at the break site and a decrease in DNA resection. Subsequently, as aging progressed, the recovery of Ku70 at DSBs decreased and the break site associated with the nuclear pore complex at the nuclear periphery, which is the location where DSB repair occurs through alternative pathways that are more mutagenic. End-bridging remained intact as HR and NHEJ declined, but eventually it too became disrupted in cells at advanced replicative age. In all, our work provides insight into the molecular changes in DSB repair pathway during replicative aging. HR first declined, resulting in a transient increase in the NHEJ. However, with increased cellular divisions, Ku70 recovery at DSBs and NHEJ subsequently declined. In wild type cells of advanced replicative age, there was a high frequency of repair products with genomic deletions and microhomologies at the break junction, events not observed in young cells which repaired primarily by HR. HighlightsO_LIDecreased DNA resection at DSBs is an early event of replicative aging C_LIO_LIEnd-joining repair increases as resection decreases at DSBs in older cells C_LIO_LIIn older cells the products of DSB repair contain deletions and microhomologies C_LIO_LIDSBs associate with the NPC at the nuclear periphery more in older cells C_LIO_LIOld Cell Enrichment method suitable for molecular biology approaches in budding yeast C_LI

molecular biology↗

bioRxiv2022

Multifunctional properties of Nej1XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice

A DNA double strand break (DSB) is primarily repaired by one of two canonical pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ requires no or minimal end processing for ligation, whereas HR requires 5 end resection followed by a search for homology. The main event that determines the mode of repair is the initiation of 5 resection because if resection starts, then NHEJ cannot occur. Nej1 is a canonical NHEJ factor that functions at the cross-roads of repair pathway choice and prior to its function in stimulating Dnl4 ligase. Nej1 competes with Dna2, inhibiting its recruitment to DSBs and thereby inhibiting resection. The highly conserved C-terminal region (CTR) of Nej1 (330-338) is important for two events that drive NHEJ, stimulating ligation and inhibiting resection, but it is dispensable for end-bridging. By combining nej1 point mutants with nuclease-dead dna2-1, we find that Nej1-F335 is essential for end-joining whereas V338 promotes NHEJ indirectly through inhibiting Dna2-mediated resection. HighlightsO_LINej1 C-terminus is critical for repair pathway choice. C_LIO_LIThe KKRK region of Nej1 is important for interactions with ssDNA and dsDNA. C_LIO_LINej1-F335 and V338 are key residues for end-joining and inhibition of resection at DSB. C_LIO_LINej1-mediated end-bridging is not sufficient for end-joining repair. C_LI

molecular biology↗

bioRxiv2021

Nej1(XLF) inhibits Sae2(CTIP)-Dna2(DNA2) mediated resection at DNA double strand breaks

The two major pathways of DNA double strand break (DSB) repair, non-homologous end-joining (NHEJ) and homologous recombination (HR), are highly conserved from yeast to mammals. The regulation of 5 DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends and more recently, it was shown to be important for DNA end-bridging and inhibiting 5 resection mediated by Dna2-Sgs1. Nej1 interacts with Sae2 and this impacts DSB repair in three ways. First, Nej1 inhibits MRX-Sae2 interactions and Sae2 localization to a DSB. Second, Nej1 inhibits Sae2-dependent recruitment of Dna2 in the absence of Sgs1. Third, NEJ1 and SAE2 showed an epistatic relationship for DNA end-bridging, an event that restrains the broken ends and reduces the frequency of genomic deletions from developing at the DSB. Deletion of NEJ1 suppressed the synthetic lethality of sae2{Delta} sgs1{Delta} and was dependent on the nuclease activity of Dna2. These Nej1 functions promote end-joining DSB repair, but could also be relevant for controlling resection initiation during HR repair. HighlightsO_LINej1 physically interacts with Sae2 and inhibits end-resection at a DSB. C_LIO_LINej1 inhibits Sae2 interactions with the MRX complex. C_LIO_LINej1 inhibits Sae2-dependent recruitment of Dna2 to a DSB. C_LIO_LINEJ1 and SAE2 are epistatic for DNA end-bridging. C_LIO_LIDeletion of NEJ1 suppresses the synthetic lethality of sae2{Delta} sgs1{Delta}, which is dependent on Dna2 nuclease activity. C_LI

genetics↗

bioRxiv2020

A Fob1 independent role for the Smc5/6 complex in the rDNA that modulates lifespan.

The ribosomal DNA (rDNA) in Saccharomyces cerevisiae is in one tandem repeat array on Chromosome XII. Two spacer regions within each repetitive element, called non-transcribed spacer 1 (NTS1) and NTS2, are important in nucleolar organization. Smc5/6 localizes to both NTS1 and NTS2 and is involved in the regulation of Sir2 and Cohibin binding at NTS1, whereas Fob1 and Sir2 are required for optimal binding of the complex to NTS1 and NTS2, respectively. We demonstrate that Smc5/6 functions in chromatin silencing at NTS1 independently of its role in homologous recombination (HR) when forks pause at the replication fork barrier (RFB). In contrast, when the complex does not localize to the rDNA in nse3-1 mutants, the shortened lifespan correlates with NTS2 homeostasis independently of FOB1 status. Our data identify the importance of Smc5/6 integrity in NTS2 transcriptional silencing and repeat tethering, which in turn underscores rDNA stability and replicative lifespan. HighlightsO_LISmc5/6 is important for transcriptional silencing in the rDNA. C_LIO_LISmc5/6 tethers the rDNA array to the periphery. C_LIO_LITranscriptional silencing of ncRNA at NTS1 and NTS2 is differentially regulated. C_LIO_LISmc5/6 has a role in rDNA maintenance independent of HR processing at the RFB. C_LIO_LIFob1-independent disruption of Smc5/6 at NTS2 leads to lifespan reduction. C_LI

molecular biology↗

bioRxiv2019

Modeling Cancer Genomic Data in Yeast Reveals Selection Against ATM Function During Tumorigenesis

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene-induced carcinogenesis. The complex is integral to all modes of double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S.cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was defective while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.\n\nAuthor SummaryA complex network of functions is required for suppressing tumorigenesis. These include processes that regulate cell growth and differentiation, processes that repair damage to DNA and thereby prevent cancer promoting mutations and signaling pathways that lead to growth arrest and programmed cell death. The Mre11 complex influences both signaling and DNA repair. To understand its role in tumor suppression, we characterized mutations affecting members of the Mre11 complex that were uncovered through cancer genomic analyses. The data reveal that the signaling functions of the Mre11 complex are important for tumor suppression to a greater degree than its role in DNA repair.

genetics↗