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Biomedical subjects

Tony R Hazbun

Publications and source records attributed to Tony R Hazbun.

7 recordsLinked to original sources
PubMed2006

Sumoylation of the budding yeast kinetochore protein Ndc10 is required for Ndc10 spindle localization and regulation of anaphase spindle elongation.

Posttranslational modification by the ubiquitin-like protein SUMO (small ubiquitin-like modifier) is emerging as an important regulator in many cellular processes, including genome integrity. In this study, we show that the kinetochore proteins Ndc10, Bir1, Ndc80, and Cep3, which mediate the attachment of chromosomes to spindle microtubules, are sumoylated substrates in budding yeast. Furthermore, we show that Ndc10, Bir1, and Cep3 but not Ndc80 are desumoylated upon exposure to nocodazole, highlighting the possibility of distinct roles for sumoylation in modulating kinetochore protein function and of a potential link between the sumoylation of kinetochore proteins and mitotic checkpoint function. We find that lysine to arginine mutations that eliminate the sumoylation of Ndc10 cause chromosome instability, mislocalization of Ndc10 from the mitotic spindle, abnormal anaphase spindles, and a loss of Bir1 sumoylation. These data suggest that sumoylation of Ndc10 and other kinetochore proteins play a critical role during the mitotic process.

Anaphase↗
PubMed2005

A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity.

Using a combination of tandem affinity purification tagging and mass spectrometry, we characterized a novel, evolutionarily conserved protein phosphatase 4 (PP4)-containing complex (PP4cs, protein phosphatase 4, cisplatin-sensitive complex) that plays a critical role in the eukaryotic DNA damage response. PP4cs is comprised of the catalytic subunit PP4C; a known regulatory subunit, PP4R2; and a novel protein that we termed PP4R3. The Saccharomyces cerevisiae PP4R3 ortholog Psy2 was identified previously in a screen for sensitivity to the DNA-damaging agent and anticancer drug cisplatin. We demonstrated that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicited cisplatin hypersensitivity. Furthermore human PP4R3 complemented the yeast psy2 deletion, and Drosophila melanogaster lacking functional PP4R3 (flfl) exhibited cisplatin hypersensitivity, suggesting a highly conserved role for PP4cs in DNA damage repair. Finally we found that PP4R3 may target PP4cs to the DNA damage repair machinery at least in part via an interaction with Rad53 (CHK2).

Cell Line↗
PubMed2004

A tethered catalysis, two-hybrid system to identify protein-protein interactions requiring post-translational modifications.

We have modified the yeast two-hybrid system to enable the detection of protein-protein interactions that require a specific post-translational modification, using the acetylation of histones and the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II as test modifications. In this tethered catalysis assay, constitutive modification of the protein to be screened for interactions is achieved by fusing it to its cognate modifying enzyme, with the physical linkage resulting in efficient catalysis. This catalysis maintains substrate modification even in the presence of antagonizing enzyme activities. A catalytically inactive mutant of the enzyme is fused to the substrate as a control such that the modification does not occur; this construct enables the rapid identification of modification-independent interactions. We identified proteins with links to chromatin functions that interact with acetylated histones, and proteins that participate in RNA polymerase II functions and in CTD phosphorylation regulation that interact preferentially with the phosphorylated CTD.

Acetylation↗
PubMed2003

Kinetochore protein interactions and their regulation by the Aurora kinase Ipl1p.

Although there has been a recent explosion in the identification of budding yeast kinetochore components, the physical interactions that underlie kinetochore function remain obscure. To better understand how kinetochores attach to microtubules and how this attachment is regulated, we sought to characterize the interactions among kinetochore proteins, especially with respect to the microtubule-binding Dam1 complex. The Dam1 complex plays a crucial role in the chromosome-spindle attachment and is a key target for phospho-regulation of this attachment by the Aurora kinase Ipl1p. To identify protein-protein interactions involving the Dam1 complex, and the effects of Dam1p phosphorylation state on these physical interactions, we conducted both a genome-wide two-hybrid screen and a series of biochemical binding assays for Dam1p. A two-hybrid screen of a library of 6000 yeast open reading frames identified nine kinetochore proteins as Dam1p-interacting partners. From 113 in vitro binding reactions involving all nine subunits of the Dam1 complex and 32 kinetochore proteins, we found at least nine interactions within the Dam1 complex and 19 potential partners for the Dam1 complex. Strikingly, we found that the Dam1p-Ndc80p and Dam1p-Spc34p interactions were weakened by mutations mimicking phosphorylation at Ipl1p sites, allowing us to formulate a model for the effects of phosphoregulation on kinetochore function.

Aurora Kinases↗
PubMed2003

Assigning function to yeast proteins by integration of technologies.

Interpreting genome sequences requires the functional analysis of thousands of predicted proteins, many of which are uncharacterized and without obvious homologs. To assess whether the roles of large sets of uncharacterized genes can be assigned by targeted application of a suite of technologies, we used four complementary protein-based methods to analyze a set of 100 uncharacterized but essential open reading frames (ORFs) of the yeast Saccharomyces cerevisiae. These proteins were subjected to affinity purification and mass spectrometry analysis to identify copurifying proteins, two-hybrid analysis to identify interacting proteins, fluorescence microscopy to localize the proteins, and structure prediction methodology to predict structural domains or identify remote homologies. Integration of the data assigned function to 48 ORFs using at least two of the Gene Ontology (GO) categories of biological process, molecular function, and cellular component; 77 ORFs were annotated by at least one method. This combination of technologies, coupled with annotation using GO, is a powerful approach to classifying genes.

Computational Biology↗
PubMed2002

A genome-wide screen for site-specific DNA-binding proteins.

We used a biochemical genomics method of assaying Saccharomyces cerevisiae proteins, derived from a nearly complete set of glutathione S-transferase fusions, to develop an approach that is able to identify proteins that bind to a DNA element. Using the upstream activation sequence (UAS) of the promoter for the invertase gene, SUC2, we identified both specific and nonspecific binding activities, which could be classified based on whether they bound with equivalent affinity to a nonspecific DNA competitor. Three transcription factors, Mig1, Yer028c, and Rgt1, were found to be binding activities specific to the SUC2 UAS. Mig1 and Yer028c had been reported previously to bind to elements within the SUC2 UAS, validating the ability of the method to identify sequence-specific factors. The third activity, Rgt1, had not been reported previously to bind to SUC2. Additional gel shift assays narrowed the Rgt1 binding site to the SUC2-B element within the SUC2 UAS, which is similar to previously identified Rgt1 binding sites present in other genes. In vivo levels of invertase activity in an rgt1Delta strain were reduced relative to an isogenic RGT+ strain when these strains were grown under inducing (low glucose) conditions, suggesting that Rgt1 may have a role in the activated transcription of SUC2. This report demonstrates the feasibility of identifying DNA binding activities by rapidly assaying a large fraction of the predicted open reading frames of an organism for binding to a regulatory DNA motif.

Binding Sites↗