ABSTRACT
Elucidating the individual and collaborative functions of genome maintenance factors is critical for understanding how genome duplication is achieved. Here, we investigate a conserved scaffold in budding yeast, Rtt107, and its three partners: a SUMO E3 complex, a ubiquitin E3 complex, and Slx4. Biochemical and genetic findings show that Rtt107 interacts separately with these partners and contributes to their individual functions, including a role in replisome sumoylation. We also provide evidence that Rtt107 associates with replisome components, and both itself and its associated E3s are important for replicating regions far from initiation sites. Corroborating these results, replication defects due to Rtt107 loss and genotoxic sensitivities in mutants of Rtt107 and its associated E3s are rescued by increasing replication initiation events through mutating two master repressors of late origins, Mrc1 and Mec1. These findings suggest that Rtt107 functions as a multi-functional platform to support replication progression with its partner E3 enzymes.
Subject(s)
DNA Replication , Endodeoxyribonucleases/genetics , Gene Expression Regulation, Fungal , Nuclear Proteins/genetics , SUMO-1 Protein/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Ubiquitin-Protein Ligases/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Endodeoxyribonucleases/metabolism , Genome, Fungal , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mutation , Nuclear Proteins/metabolism , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , SUMO-1 Protein/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sumoylation , Ubiquitin-Protein Ligases/metabolismABSTRACT
Gastrointestinal stromal tumors (GIST) are the most common type of soft tissue sarcoma that occur throughout the gastrointestinal tract. Most of these tumors are caused by oncogenic activating mutations in the KIT or PDGFRA genes. The NCCN Guidelines for GIST provide recommendations for the diagnosis, evaluation, treatment, and follow-up of patients with these tumors. These NCCN Guidelines Insights summarize the panel discussion behind recent important updates to the guidelines, including revised systemic therapy options for unresectable, progressive, or metastatic GIST based on mutational status, and updated recommendations for the management of GIST that develop resistance to specific tyrosine kinase inhibitors.
Subject(s)
Gastrointestinal Stromal Tumors , Humans , Gastrointestinal Stromal Tumors/diagnosis , Gastrointestinal Stromal Tumors/genetics , Gastrointestinal Stromal Tumors/therapy , Receptor, Platelet-Derived Growth Factor alpha/genetics , Proto-Oncogene Proteins c-kit/genetics , MutationABSTRACT
Soft tissue sarcomas (STS) are rare malignancies of mesenchymal cell origin that display a heterogenous mix of clinical and pathologic characteristics. STS can develop from fat, muscle, nerves, blood vessels, and other connective tissues. The evaluation and treatment of patients with STS requires a multidisciplinary team with demonstrated expertise in the management of these tumors. The complete NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Soft Tissue Sarcoma provide recommendations for the diagnosis, evaluation, and treatment of extremity/superficial trunk/head and neck STS, as well as retroperitoneal/intra-abdominal STS, desmoid tumors, and rhabdomyosarcoma. This portion of the NCCN Guidelines discusses general principles for the diagnosis and treatment of retroperitoneal/intra-abdominal STS, outlines treatment recommendations, and reviews the evidence to support the guidelines recommendations.
Subject(s)
Sarcoma , Soft Tissue Neoplasms , Extremities/pathology , Humans , Medical Oncology , Sarcoma/drug therapy , Sarcoma/therapy , Soft Tissue Neoplasms/diagnosis , Soft Tissue Neoplasms/pathology , Soft Tissue Neoplasms/therapyABSTRACT
The cellular response to DNA damage employs multiple dynamic protein modifications to exert rapid and adaptable effects. Substantial work has detailed the roles of canonical checkpoint-mediated phosphorylation in this program. Recent studies have also implicated sumoylation in the DNA damage response; however, a systematic view of the contribution of sumoylation to replication and repair and its interplay with checkpoints is lacking. Here, using a biochemical screen in yeast, we establish that DNA damage-induced sumoylation occurs on a large scale. We identify MRX (Mre11-Rad50-Xrs2) as a positive regulator of this induction for a subset of repair targets. In addition, we find that defective sumoylation results in failure to complete replication of a damaged genome and impaired DNA end processing, highlighting the importance of the SUMO-mediated response in genome integrity. We also show that DNA damage-induced sumoylation does not require Mec1 checkpoint signaling, and the presence of both enables optimal DNA damage resistance.
Subject(s)
DNA Repair Enzymes/metabolism , DNA Repair , DNA Replication , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , Sumoylation , Cell Cycle Checkpoints , DNA Damage , DNA-Binding Proteins/metabolism , Gene Knockout Techniques , Genome, Fungal , Genomic Instability , Intracellular Signaling Peptides and Proteins/genetics , Microbial Viability , Multiprotein Complexes/metabolism , Phosphorylation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The Ku70-Ku80 ring complex encloses DNA ends to facilitate telomere maintenance and DNA break repair. Many studies focus on the ring-forming regions of subunits Ku70 and Ku80. Less is known about the Ku70 C-terminal tail, which lies outside the ring. Our results suggest that this region is responsible for dynamic sumoylation of Yku70 upon DNA association in budding yeast. Mutating a cluster of five lysines in this region largely eliminates Yku70 sumoylation. Chromatin immunoprecipitation analyses show that yku70 mutants with these lysines replaced by arginines exhibit reduced Ku-DNA association at both telomeres and internal DNA breaks. Consistent with this physical evidence, Yku70 sumoylation deficiency is associated with impaired ability to block DNA end resection and suppression of multiple defects caused by inefficient resection. Correlating with these, yku70 mutants with reduced sumoylation levels exhibit shorter telomeres, increased G overhang levels, and altered levels of non-homologous end joining. We also show that diminution of sumoylation does not affect Yku70 protein levels or its interactions with protein and RNA partners. These results suggest a model whereby Yku70 sumoylation upon DNA association strengthens Ku-DNA interaction to promote multiple functions of Ku.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , DNA End-Joining Repair , DNA Repair , Gene Expression Regulation, Fungal , Lysine/chemistry , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phenotype , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , Saccharomyces cerevisiae/metabolism , Sumoylation , Telomere/ultrastructureABSTRACT
Protein-protein interactions mediate essential cellular processes, however the detection of native interactions is challenging since they are often low affinity and context dependent. Here, we develop a chemical proteomics approach in vivo CLASPI [iCLASPI] (in vivo crosslinking-assisted and stable isotope labeling by amino acids in cell culture [SILAC]-based protein identification) relying upon photo-crosslinking, amber suppression, and SILAC-based quantitative proteomics to profile context-dependent protein-protein interactions in living cells. First, we use iCLASPI to profile in vivo binding partners of the N-terminal tails of soluble histone H3 or H4. We identify known histone chaperones and modifying proteins, thereby validating our approach, and find an interaction between soluble histone H3 and UBR7, an E3 ubiquitin ligase, mediated by UBR7's PHD domain. Furthermore, we apply iCLASPI to profile the context-dependent protein-protein interactions of chromatin-associated histone H3 at different cell-cycle stages, and identify ANP32A as a mitosis-specific interactor. Our results demonstrate that the iCLASPI approach can provide a general strategy for identifying native, context-dependent direct protein-protein interactions using photo-crosslinking and quantitative proteomics.
Subject(s)
Chromatin/chemistry , Proteomics , Ubiquitin-Protein Ligases/chemistry , Amino Acids/chemistry , Amino Acids/metabolism , Animals , Cell Culture Techniques , Cell Cycle , Cell Line , Chromatin/metabolism , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/metabolism , HEK293 Cells , Histones/chemistry , Histones/metabolism , Humans , Isotope Labeling , Mice , Protein Binding , Ubiquitin-Protein Ligases/metabolismABSTRACT
Genome maintenance requires coordinated actions of diverse DNA metabolism processes. Scaffolding proteins, such as those containing multiple BRCT domains, can influence these processes by collaborating with numerous partners. The best-studied examples of multi-BRCT scaffolds are the budding yeast Dpb11 and its homologues in other organisms, which regulate DNA replication, repair, and damage checkpoints. Recent studies have shed light on another group of multi-BRCT scaffolds, including Rtt107 in budding yeast and related proteins in other organisms. These proteins also influence several DNA metabolism pathways, though they use strategies unlike those employed by the Dpb11 family of proteins. Yet, at the same time, these 2 classes of multi-BRCT proteins can collaborate under specific situations. This review summarizes recent advances in our understanding of how these multi-BRCT proteins function in distinct manners and how they collaborate, with a focus on Dpb11 and Rtt107.
Subject(s)
Genome, Fungal , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Cell Cycle Checkpoints/genetics , Recombinational DNA Repair/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
Corn silage juice was found to be a favorable substrate for production of fodder yeasts. Kluyveromyces marxianus NRRL Y-610 yielded significantly more cell dry weight than other cultures examined. In shake-flask experiments, the yeast produced over 13 g of cell dry weight per liter of corn silage juice and completely consumed the organic pollutants (lactic acid, acetic acid, and ethanol). The yeast settled rapidly and had a yeast volume index of 21 ml/g. The results indicate that K. marxianus NRRL Y-610 could be used to efficiently remove lactic acid and other organic compounds from corn silage juice with the concomitant production of fodder yeast.
Subject(s)
Acetic Acid/metabolism , Ethanol/metabolism , Kluyveromyces/physiology , Lactic Acid/metabolism , Zea mays , Acetic Acid/isolation & purification , Animal Feed , Biomass , Ethanol/isolation & purification , Fermentation , Lactic Acid/isolation & purificationABSTRACT
The Smc5-Smc6 complex in Saccharomyces cerevisiae is both essential for growth and important for coping with genotoxic stress. While it facilitates damage tolerance throughout the genome under genotoxin treatment, its function during unperturbed growth is mainly documented for repetitive DNA sequence maintenance. Here we provide physical and genetic evidence showing that the Smc5-Smc6 complex regulates recombination at non-repetitive loci such as centromeres in the absence of DNA damaging agents. Mutating Smc6 results in the accumulation of recombination intermediates at centromeres and other unique sequences as assayed by 2D gel analysis. In addition, smc6 mutant cells exhibit increased levels of Rad52 foci that co-localize with centromere markers. A rad52 mutation that decreases centromeric, but not overall, levels of Rad52 foci in smc6 mutants suppresses the nocodazole sensitivity of these cells, suggesting that the Smc6-mediated regulation of recombination at centromeric regions impacts centromere-related functions. In addition to influencing recombination, the SUMO ligase subunit of the Smc5-Smc6 complex promotes the sumoylation of two kinetochore proteins and affects mitotic spindles. These results suggest that the Smc5-Smc6 complex regulates both recombination and kinetochore sumoylation to facilitate chromosomal maintenance during growth.
Subject(s)
Cell Cycle Proteins/metabolism , Centromere/physiology , Kinetochores/metabolism , Recombination, Genetic , SUMO-1 Protein/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/growth & development , Chromosomes, Fungal/genetics , DNA Damage , DNA Repair , Electrophoresis, Gel, Two-Dimensional , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spindle Apparatus , SumoylationABSTRACT
Telomere length homeostasis is an important aspect of telomere biology. Here, we show that SUMOylation limits telomere length and targets multiple telomere proteins in Saccharomyces cerevisiae. A main target is Cdc13, which both positively and negatively regulates telomerase and confers end protection. We demonstrate that Cdc13 SUMOylation restrains telomerase functions by promoting Cdc13 interaction with the telomerase inhibitor Stn1 without affecting end protection. Mutation of the Cdc13 SUMOylation site (cdc13-snm) lengthens telomeres and reduces the Stn1 interaction, whereas Cdc13-SUMO fusion has the opposite effects. cdc13-snm's effect on telomere length is epistatic with stn1, but not with yku70, tel1 or est1 alleles, and is suppressed by Stn1 overexpression. Cdc13 SUMOylation peaks in early-mid S phase, prior to its known Cdk1-mediated phosphorylation, and the two modifications act antagonistically, suggesting that the opposite roles of Cdc13 in telomerase regulation can be separated temporally and regulated by distinct modifications.