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

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