DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase

DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center. Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine (2AP) to measure the rates of active site switching for the bacteriophage T4 DNA polymerase. There are three findings: (i) the rate of return of the trimmed primer-end from the exonuclease to the polymerase active center is rapid, >500 s−1; (ii) T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonuclease-to-polymerase and polymerase-to-exonuclease active site switching steps and (iii) proofreading reactions that initiate in the polymerase active center are not intrinsically processive

The Impact of Cyclin B1 on Tuberin Stabilization

Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder caused by various mutations in either TSC1 or TSC2, genes that encode the proteins Hamartin and Tuberin respectively. Hamartomas (benign tumours), skin lesions, neurological symptoms, renal dysfunctions, and retinal malformations are often present in TSC patients with varying severity. Tuberin and Hamartin regulate protein synthesis through mTORC1 inhibition. Tuberin can also delay mitotic onset at the G2/M cell cycle transition by binding to Cyclin B1. Hamartin has been shown to stabilize Tuberin by inhibiting its ubiquitination by HERC1 and subsequent degradation. Preliminary data from our lab suggests that Cyclin B1 may also contribute to the stabilization of Tuberin levels during the G2/M transition. Phosphorylation status of the cytoplasmic retention sequence (CRS) of Cyclin B1 plays an important role in the formation of the Tuberin/Cyclin B1 complex. The unphosphorylated CRS form of Cyclin B1 (Cyclin B1 5xA) binds stronger to Tuberin compared to the phosphorylated form (Cyclin B1 5xE). My thesis investigates the role of Cyclin B1 in Tuberin stabilization and how CRS phosphorylation status impacts Tuberin/Cyclin B1 complex formation. HEK293-TSC1 null cells (IC2) will be transfected with varying concentrations of Cyclin 5xA DNA and the levels of the Tuberin protein will be quantified by Western blot techniques. The importance of each residue in the CRS region for the Tuberin/Cyclin B1 complex formation will also be evaluated using Immunoprecipitation studies. Understanding the role of Cyclin B1 in Tuberin stabilization will shed light on cell proliferation and growth mechanisms that underlie tumorigenic disorders

The Role of Tuberin in DNA Damage Repair During Cell Proliferation

The cell cycle contains DNA damage checkpoints that delay mitotic progression and allow for DNA repair before cell division. DNA damage can be caused by radiation, drugs, and other processes which lead to cellular mutations and carcinogenesis. The tumour suppressor protein p53 is activated in the presence of DNA damage. It induces apoptosis or cell cycle arrest which allows cells to repair themselves. Tuberin (TSC2), another tumour suppressor protein, regulates the G2/M transition in the cell cycle and negatively regulates protein synthesis and cell growth. Mutations in tuberin can lead to the multisystem autosomal dominant disease known as tuberous sclerosis (TSC). Previously, our lab has shown that Tuberin regulates mitotic onset through cellular localization of the G2/M Cyclin, Cyclin B1. My project focuses on the Tuberin/Cyclin B1 complex in relation to G2/M arrest and DNA damage repair. In this study, we will overexpress Tuberin-WT and Tuberin clinical mutants in NIH-3T3 (mouse) and U2OS (human) p53 wild type cells. Etoposide, a topoisomerase II drug, will be used to induce DNA damage. Cells will then be analyzed by flow cytometry, TUNEL assay, and western blot to assess their cell cycle profile, apoptotic levels, and protein expression. Using CRISPR-Cas9 technology, a NIH-3T3 null TSC2 cell line will be created to clarify the role of Tuberin during DNA repair. Preliminary results have determined that 4μM of etoposide treatment at 8 hours arrests 50% of NIH-3T3 cells at G2/M. This project will provide greater insight into DNA damage induced carcinogenesis, TSC, and other proliferative diseases

Applying CRISPR/Cas9 and fluorescent tools to dissect the role of Tuberin in cell cycle regulation

How cells regulate their growth and division involves a tightly controlled integration of many mechanisms. In cells, Tuberin (gene – TSC2) is a protein in the Tuberous Sclerosis Complex (TSC) that modulates cellular growth, size, and proliferation. Mutations in the proteins forming the TSC can cause Tuberous Sclerosis Complex, an autosomal dominant disorder characterized by multisystem pathologies that is often associated with benign hamartomas in the brain, kidney, lungs and skin. The focus of my research is to clarify the role of Tuberin in the regulation of cell size and proliferation at the G2/M cell cycle checkpoint. During late G2, Tuberin retains Cyclin B1 (gene – CCNB1), a mitotic cyclin, in the cytoplasm thereby prolonging mitotic onset. We constructed six TSC2 mutants that harbour clinically relevant mutations which are known to destabilize the TSC. Interestingly, these mutations fall within the Tuberin Cyclin B1 binding domain. Whether or not these mutations disrupt the regulation of the G2/M checkpoint is a key question of this project. This is studied by over-expressing the mutants with GFP tagged Cyclin B1 in Tuberin null cells. The resultant phenotypes are analyzed by flow cytometry, immunoprecipitation, and immunofluorescence. To aid in the temporal study of the cell cycle, I aim to validate successful CRISPR/Cas9-mediated knock-in of an iRFP tag within the TSC2 gene of HEK293 cells. This new cell line will be a powerful tool to dissect the roles of Tuberin in regulating cellular growth and division and can provide deep understanding of proliferative diseases like TSC and cancers

Cell Size regulation by a New Cell Cycle checkpoint: Characterization of clinically relevant Tuberin mutants

Tuberous sclerosis (TS) is a multi-system genetic disease caused by the growth of benign tumours primarily in the brain, kidneys, heart, eyes, lungs, and skin. TS has particularly severe consequences on the central nervous system, resulting in seizures, developmental delay and behavioral problems. This disorder affects around 1.5 million individuals worldwide and occurs by a mutation in one of two genes; TSC1 or TSC2. The TSC2 gene encodes for the protein Tuberin, a tumour suppressor protein well known for it’s ability to regulated cell growth and the cell cycle. Altered levels of Tuberin and mutations in this protein have been found in several cancers, including medulloblastoma and skin cancer. We have established that Tuberin binds and regulates the G2/M cyclin, Cyclin B1 (CycB1) creating a new G2/M checkpoint. Our results show that the Tuberin/CycB1 interaction regulates cell size and this regulation is nutrient dependent. Several mutations responsible for TS are present in the CycB1 binding domain located in the N-terminal domain of Tuberin. It is our hypothesis that these mutations can affect the Tuberin/CycB1 interaction and result in dysregulation of cell proliferation and cell size. Using site-directed mutagenesis we constructed six TSC2 mutants to study the phenotypes in HEK293 and NIH3T3 cells. We have demonstrated that one mutation, Tuberin-C698Y, has lower affinity for CycB1 binding and presents a nuclear localization instead of the usual cytoplasmic localization of the wild type complex. We are focusing on this mutation to determine the full range of consequences of abrogating this interaction. Importantly, we are inserting the Tuberin-C698Y mutation into the HEK293 cells genome through the CRISPR-Cas9 system to determine the endogenous significance of this specific change. The phenotype of these cells will be studied by immunofluorescence and flow cytometry techniques. Patients with specific TSC2 mutations develop TS and have an increased chance of select cancers. Having a better understanding of how specific changes in this large protein alters fundamental cell biology such as cell proliferation and cell size can ultimately help to effectively treat patients with these specific mutations

Derivation of a Novel G2 Reporter System

Abstract Progression through G2 phase of the cell cycle is a technically difficult area of cell biology to study due to the lack of physical markers specific to this phase. The FUCCI system uses the biology of the cell cycle to drive fluorescence in select phases of the cell cycle. Similarly, a commercially available system has used a fluorescent analog of the Cyclin B1 protein to visualize cells from late S phase to the metaphase– anaphase transition. We have modified these systems to use the promoter and destruction box elements of Cyclin B1 to drive a cyan fluorescent protein. We demonstrate here that this is a useful tool for measuring the length of G2 phase without perturbing any aspect of cell cycle progression

How Does Cyclin B1 Influence Cell Size

The tumour suppressor protein, Tuberin is implicated in controlling both growth and division of the cell. Growth is controlled by integrating various nutrient sensing pathways to inhibit protein synthesis and therefore, cell size. Our lab has found that Tuberin controls division by binding to and regulating the localization of the G2/M cyclin, Cyclin B1. Preliminary results show that manipulation of Cyclin B1 and Tuberin results in increased Tuberin protein levels. Therefore, I would like to determine how Cyclin B1 affects the biology of the Tuberin protein. My first aim is to elucidate the residues on Cyclin B1 that mediate Tuberin binding. Cyclin B1 phospho-mutants will be created to determine which residues result in abrogation of Tuberin-Cyclin B1 binding. My second aim is to determine the effect of Cyclin B1 binding on Tuberin. The mutants created in aim one will be used to determine the effect of disrupting Tuberin-Cyclin B1 binding on the protein levels of Tuberin. In addition, the proteasome and lysosome will be inhibited to determine what mechanism Cyclin B1 uses to increase Tuberin protein levels. Lastly, I will also use CRISPR-Cas9 to create a fluorescent Tuberin cell line and use live cell microscopy to monitor Tuberin degradation as it progresses through the cell cycle. How a cell balances growth and division is crucial towards understanding cell size regulation

The Role of Tuberin and Cyclin B1 as a DNA Damage Response during the G2/M Transition

Tuberous Sclerosis (TS) is a multi-system disorder that causes the formation of benign tumours called hamartomas. In rare cases this disease can progress to aggressive cancers. The cause of TS is a mutation in either the TSC1 or TSC2 gene that encode for the tumour suppressor proteins Hamartin or Tuberin protein, respectively. Dr. Porter’s lab has characterized how Tuberin is able to regulate the G2/M transition of the cell cycle by binding and regulating the localization of CyclinB1, the protein that allows for the transition of the cell into mitosis. The hypothesis is that misregulation of this process may facilitate the accumulation of damaged DNA allowing for progression to malignancy. This thesis answers the following questions: What are the sites on CyclinB1 that are important in mediating the interaction between Tuberin? Does interaction with CyclinB1 stabilize the Tuberin protein? Does altered binding result in the accumulation of DNA damage? These questions will be assessed with 3 aims: 1) CyclinB1 mutants was constructed and binding in HEK293 has been measured using immunoprecipitation and western blotting techniques. 2) The stability of Tuberin was tested with graded amounts of CyclinB1 and measured using western blotting techniques. 3) The accumulation of DNA damage was tested with wild-type or mutated forms of Tuberin with reduced binding to CyclinB1. The data demonstrates that post-translational modifications of CyclinB1 are critical for mediating the interaction with Tuberin and that abrogation of this binding results in reduced protein levels of Tuberin and enhanced accumulation of DNA damage. These findings support that the Tuberin-CyclinB1 interaction is an important cellular checkpoint that protects cell integrity. Dissecting the mechanics of this checkpoint may reveal novel mechanisms for treating select benign and malignant tumours that impact the lives of many Canadians annually, with the majority of these being pediatric cases

The Tumour Suppressor Tuberin Coordinating DNA damage and Mitotic Onset.

The cell cycle is a series of events that involve cell growth and division. It is regulated by Cyclins and CDKs, as well as checkpoints that control its progression. Tuberin (TSC2 gene) is a tumour suppressor protein that controls cell growth and cell proliferation. Tuberin mutations are implicated in several cancers and diseases such as tuberous sclerosis complex (TSC), a multisystem genetic disorder that is characterized by the growth of hamartomas. Previous data from our lab has demonstrated Tuberin’s role in controlling miotic onset by binding to the G2/M cyclin, Cyclin B1 according to nutrient availability. My project explores the role of the tuberin in the G2/M transition during DNA damage repair. DNA damage can result from events such as radiation, X-ray, and reactive oxygen species which activates the G2/M checkpoint to restrict mitotic onset and ensure sufficient time to repair damaged DNA. In our studies, NIH3T3 p53 positive cell line cells are being transfected with TSC2-WT or clinical TSC2 mutants that present low affinity for Cyclin B1. The transfected cells will be treated with etoposide, a topoisomerase II drug that induces double-stranded DNA breaks during the G2/M transition. The changes in cell cycle phases are being analyzed by flow cytometry. Our preliminary results have determined the best concentration of etoposide (10uM) and timing (4 hours) to promote G2/M arrest in our experimental conditions. This project will help expand the role of Tuberin during mitotic onset and clarify mechanisms of proliferative diseases as TSC and cancers