Recruiting a Microtubule-Binding Complex to DNA Directs Chromosome Segregation in Budding Yeast

Accurate chromosome segregation depends on the kinetochore, the complex of proteins that link microtubules to centromeric DNA1. The budding yeast kinetochore consists of more than 80 proteins assembled on a 125bp region of DNA1. We studied the assembly and function of kinetochore components by fusing individual kinetochore proteins to the lactose repressor (LacI) and testing their ability to improve the segregation of a plasmid carrying tandem repeats of the lactose operator (LacO). Targeting Ask1, a member of the Dam1/DASH microtubule-binding complex, creates a synthetic kinetochore that performs many functions of a natural kinetochore: it can replace an endogenous kinetochore on a chromosome, biorient sister kinetochores at metaphase of mitosis, segregate sister chromatids, and repair errors in chromosome attachment. We show the synthetic kinetochore’s functions do not depend on the DNA-binding components of the natural kinetochore but do require other kinetochore proteins. We conclude that tethering a single kinetochore protein to DNA triggers the assembly of the complex structure that directs mitotic chromosome segregation.Molecular and Cellular Biolog

Localization and Function of Budding Yeast CENP-A Depends upon Kinetochore Protein Interactions and Is Independent of Canonical Centromere Sequence

SummaryIn many eukaryotes, the centromere is epigenetically specified and not strictly defined by sequence. In contrast, budding yeast has a specific 125 bp sequence required for kinetochore function. Despite the difference in centromere specification, budding yeast and multicellular eukaryotic centromeres contain a highly conserved histone H3 variant, CENP-A. The localization of budding yeast CENP-A, Cse4, requires the centromere DNA binding components, which are not conserved in multicellular eukaryotes. Here, we report that Cse4 localizes and functions at a synthetic kinetochore assembly site that lacks centromere sequence. The outer kinetochore Dam1-DASH and inner kinetochore CBF3 complexes are required for Cse4 localization to that site. Furthermore, the natural kinetochore also requires the outer kinetochore proteins for full Cse4 localization. Our results suggest that Cse4 localization at a functional kinetochore does not require the recognition of a specific DNA sequence by the CBF3 complex; rather, its localization depends on stable interactions among kinetochore proteins

Nondisjunction of a Single Chromosome Leads to Breakage and Activation of DNA Damage Checkpoint in G2

The resolution of chromosomes during anaphase is a key step in mitosis. Failure to disjoin chromatids compromises the fidelity of chromosome inheritance and generates aneuploidy and chromosome rearrangements, conditions linked to cancer development. Inactivation of topoisomerase II, condensin, or separase leads to gross chromosome nondisjunction. However, the fate of cells when one or a few chromosomes fail to separate has not been determined. Here, we describe a genetic system to induce mitotic progression in the presence of nondisjunction in yeast chromosome XII right arm (cXIIr), which allows the characterisation of the cellular fate of the progeny. Surprisingly, we find that the execution of karyokinesis and cytokinesis is timely and produces severing of cXIIr on or near the repetitive ribosomal gene array. Consequently, one end of the broken chromatid finishes up in each of the new daughter cells, generating a novel type of one-ended double-strand break. Importantly, both daughter cells enter a new cycle and the damage is not detected until the next G2, when cells arrest in a Rad9-dependent manner. Cytologically, we observed the accumulation of damage foci containing RPA/Rad52 proteins but failed to detect Mre11, indicating that cells attempt to repair both chromosome arms through a MRX-independent recombinational pathway. Finally, we analysed several surviving colonies arising after just one cell cycle with cXIIr nondisjunction. We found that aberrant forms of the chromosome were recovered, especially when RAD52 was deleted. Our results demonstrate that, in yeast cells, the Rad9-DNA damage checkpoint plays an important role responding to compromised genome integrity caused by mitotic nondisjunction

Normal microtubule function and the interaction between the pathways for tubulin folding and expression in S. cerevisiae

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2003.Includes bibliographical references.Undimerized -tubulin is toxic to the yeast Saccharomyces cereivisiae. Free P-tubulin can arise if the tubulin heterodimer dissociates or if levels of 3-tubulin and cc-tubulin are unbalanced. I am using the toxicity of 3-tubulin to understand the early steps in microtubule morphogenesis. I have found that a mutation of the gene PLP1 allows cells to survive the toxicity of [3-tubulin produced from disparate levels of - and -tubulin. The suppression occurs either when c-tubulin is modestly underexpressed relative to -tubulin, or when 13-tubulin is inducibly and strongly overexpressed. A significant proportion of the undimerized -tubulin in plpl cells is less toxic and less functional than in wild type cells. As a result, piplA cells have lower levels of heterodimer. Significantly, plpl cells that also lack PaclOp, a component of the GimC/Pfd complex that helps fold tubulin polypeptides, are even less affected by free -tubulin. Our results suggest that Plplp defines a novel step in -tubulin folding. My work demonstrates an interaction between the pathways for tubulin folding and the regulation of tubulin expression. Cells that are paclOA piplA have much less folded and functional -tubulin than even plplA cells, and also upregulate -tubulin through increasing transcription. The upregulation of -tubulin RNA is dependent on the putative transcription factor Cin5p. In the absence of CIN5, paclO plpl A cells have decreased tubulin heterodimer levels, down to approximately 20% that of wild type. The heterodimer levels are also decreased from paclO plpl cells suggesting that the limiting factor in heterodimer formation in paclO plpl in5A is -tubulin. The paclOA plpl cin5 cells grow normally, but have mitotic defects such as abnormal nuclear positioning and short anaphase spindles.(cont.) The Cin5p dependent upregulation of 3-tubulin may be a mechanism to maintain tubulin heterodimer levels and so sustain normal microtubule function.by Soni Lacefield Shimoda.Ph.D

WHI3 Regulation of CDK in S. cerevisiae.

There are many diseases associated with malfunc4ons in the cell cycle. For instance, aneuploidy—when daughter cells have the abnormal number of chromosomes—results from improper cell division. Diseases that arise from chromosomal abnormali4es range from Down Syndrome to Turner Syndrome to Patau Syndrome, all extremely debilita4ng afflic4ons. Another serious consequence of unregulated cell division is the development of cancers. One of the hallmarks of cancer cells is cell prolifera4on, which is a result of unregulated cell division. Studying cell cycle regula4on in yeast, par4cularly budding yeast, Saccharomyces cerevisiae, allows for a beFer understanding of the human cell cycle. Many of the genes studied in my lab are conserved in humans, meaning that those yeast cell proteins also func4on in human cells. Mito4c cell division—the type of division in which a mother cell produces two iden4cal daughter cells—is regulated by a protein complex called cyclin-dependent kinase (CDK). This protein complex has been intensely studied by cell biologists, yet there is s4ll much that is unknown about how it is controlled. CDK—which must be ac4ve in order for cell division to occur—is regulated by a protein in yeast called Swe1. Swe1 inhibits CDK when the cell is perturbed, thereby hal4ng cell division. One way that the cell can be perturbed is by the dele4on of the protein ELM1. When present, ELM1 regulates the cytoskeleton of the cell. However, when ELM1 is deleted, Swe1 is ac4vated, which results in a delayed cell division and irregularly long buds. These long buds are also the result of sep4n perturba4on. When func4oning properly, sep4n proteins form a ring around the bud neck. We recently found that the dele4on of another protein called WHI3 rescues the ELM1 dele4on-induced cell division delay and long buds. By u4lizing fluorescent microscopy, we have been able to visualize cells lacking both WHI3 and ELM1. Our hypothesis is that WHI3 is somehow involved in regula4on of CDK. In order to test this hypothesis, we are working on experiments to see what happens to CDK and other regulators of cell division when ELM1 is deleted, WHI3 is deleted, and when both WHI3 and ELM1 are deleted. I will be conduc4ng other molecular biology experiments to measure the level of CDK ac4vity in both the cytoplasm and the nucleus. My work will help to elucidate another mechanism by which CDK is regulated, which will contribute to our overall understanding of proper progression through the cell cycle

Meiotic cells escape prolonged spindle checkpoint activity through kinetochore silencing and slippage.

To prevent chromosome mis-segregation, a surveillance mechanism known as the spindle checkpoint delays the cell cycle if kinetochores are not attached to spindle microtubules, allowing the cell additional time to correct improper attachments. During spindle checkpoint activation, checkpoint proteins bind the unattached kinetochore and send a diffusible signal to inhibit the anaphase promoting complex/cyclosome (APC/C). Previous work has shown that mitotic cells with depolymerized microtubules can escape prolonged spindle checkpoint activation in a process called mitotic slippage. During slippage, spindle checkpoint proteins bind unattached kinetochores, but the cells cannot maintain the checkpoint arrest. We asked if meiotic cells had as robust of a spindle checkpoint response as mitotic cells and whether they also undergo slippage after prolonged spindle checkpoint activity. We performed a direct comparison between mitotic and meiotic budding yeast cells that signal the spindle checkpoint through two different assays. We find that the spindle checkpoint delay is shorter in meiosis I or meiosis II compared to mitosis, overcoming a checkpoint arrest approximately 150 minutes earlier in meiosis than in mitosis. In addition, cells in meiosis I escape spindle checkpoint signaling using two mechanisms, silencing the checkpoint at the kinetochore and through slippage. We propose that meiotic cells undertake developmentally-regulated mechanisms to prevent persistent spindle checkpoint activity to ensure the production of gametes

WHI3 Regulation of Cyclin-Dependent Kinase Activity in Saccharomyces cerevisiae

There are many diseases associated with malfunctions in the cell cycle. For instance, aneuploidy—when daughter cells have the abnormal number of chromosomes—results from improper cell division. Diseases that arise from chromosomal abnormalities range from Down Syndrome to Turner Syndrome to Patau Syndrome, all extremely debilitating afflictions. Another serious consequence of unregulated cell division is the development of cancers. One of the hallmarks of cancer cells is cell proliferation, which is a result of unregulated cell division. Studying cell cycle regulation in yeast, particularly budding yeast, Saccharomyces cerevisiae, allows for a better understanding of the human cell cycle. Many of the genes studied in my lab are conserved in humans, meaning that those yeast cell proteins also func4on in human cells. Mitotic cell division—the type of division in which a mother cell produces two identical daughter cells—is regulated by a protein complex called cyclin-dependent kinase (CDK). This protein complex has been intensely studied by cell biologists, yet there is s4ll much that is unknown about how it is controlled. CDK—which must be active in order for cell division to occur—is regulated by a protein in yeast called Swe1. Swe1 inhibits CDK when the cell is perturbed, thereby halting cell division. One way that the cell can be perturbed is by the dele4on of the protein ELM1. When present, ELM1 regulates the cytoskeleton of the cell. However, when ELM1 is deleted, Swe1 is activated, which results in a delayed cell division and irregularly long buds. These long buds are also the result of sep4n perturbation. When functioning properly, sep4n proteins form a ring around the bud neck. We recently found that the dele4on of another protein called WHI3 rescues the ELM1 dele4on-induced cell division delay and long buds. By utilizing fluorescent microscopy, we have been able to visualize cells lacking both WHI3 and ELM1. Our hypothesis is that WHI3 is somehow involved in regulation of CDK. In order to test this hypothesis, we are working on experiments to see what happens to CDK and other regulators of cell division when ELM1 is deleted, WHI3 is deleted, and when both WHI3 and ELM1 are deleted. I will be conducting other molecular biology experiments to measure the level of CDK activity in both the cytoplasm and the nucleus. My work will help to elucidate another mechanism by which CDK is regulated, which will contribute to our overall understanding of proper progression through the cell cycle