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

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

The tumor suppressor tuberin regulates mitotic onset through the cellular localization of cyclin B1

Tuberous sclerosis is a multi-organ disorder characterized by the formation of benign tumors, called hamartomas, which affect more than 1 million people worldwide. The syndrome is initiated by a mutation in one of two tumor suppressor genes, TSC1 or TSC2 which encode for the proteins hamartin and tuberin, respectively. Herein we demonstrate that tuberin binds and regulates the G(2)/M cyclin, cyclin B1. We have determined that this binding region encompasses a mutational hotspot within tuberin implicated in some of the most severe cases of TS. Mimicking a mutation found in a subset of patients with Tuberous sclerosis we found a significant reduction in the binding between tuberin and cyclin B1. Functionally, our data supports that tuberin plays a role in regulating the cellular localization of cyclin B1. These results demonstrate a novel and clinically relevant mechanism where tuberin functions in mitotic onset

The Cyclin-like Protein Spy1 Regulates Growth and Division Characteristics of the CD133+ Population in Human Glioma

Summary The heterogeneity of brain cancers, as most solid tumors, complicates diagnosis and treatment. Identifying and targeting populations of cells driving tumorigenesis is a top priority for the cancer biology field. This is not a trivial task; considerable variance exists in the driving mutations, identifying markers, and evolutionary pressures influencing initiating cells in different individual tumors. Despite this, the ability to self-renew and differentiate must be conserved to reseed a heterogeneous tumor mass. Focusing on one example of a tumor-initiating cell population, we demonstrate that the atypical cyclin-like protein Spy1 plays a role in balancing the division properties of glioma cells with stemness properties. This mechanistic insight may provide new opportunities for therapeutic intervention of brain cancer