DNA profiling of exhibits that originate from forensic stock theft cases is routinely used as a tool to link suspects to the crime or scene. DNA derived from aged or degraded samples is often highly fragmented which compromises the efficiency for obtaining a complete genotypic profile using PCR. Conventional polymerases such as Taq, lack certain repair mechanisms for use on degraded DNA templates. New generation polymerases are known to have high fidelity characteristics. The aim of this study was to determine the efficiency of Restorase®, a novel DNA polymerase blend that is known to repair damaged DNA and the FastStart High Fidelity PCR System enzymes, on degraded forensic bovine samples using PCR-based methodology. Bovine meat samples were subjected to different degrees of degradation in the sun and in the shade during summer and winter seasons. DNA was extracted, subjected to PCR amplification using 16 bovine microsatellites and genotypes were generated for analyses. Rapid degradation of samples was observed during winter while during summer samples tend to dry out. Restorase® exhibited high enzyme activity on degraded samples as compared with FastStart and Taq DNA polymerase. Some of the markers that failed to be successfully amplified by Taq polymerase, such as ETH10 and SPS115 were recovered using Restorase®. Markers such as BM1818, BM2113, ETH3, INRA23 and TGLA227 remained active throughout the experiment using all the enzymes, and therefore can form a basis of the bovine marker panel. Restorase® was found to be an alternative enzyme for use in bovine forensic analysis. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Animal and Wildlife Sciences / unrestricted

DNA replication is a fundamental process in all living organisms. It is a semi- discontinuous process in which the leading strand is synthesized continuously and the lagging strand is synthesized discontinuously as short Okazaki fragments (OF). The initiation of DNA synthesis requires DNA polymerase α (Pol α/primase) in complex with the primase to form a complex of four subunits. Pol α/primase is the only enzyme that can perform de novo DNA synthesis on single-stranded DNA. The catalytic subunit of the primase (PRIM1) synthesizes RNA primers that are approximately nine nucleotides long. The synthesized RNA primers are then passed intramolecularly to the polymerase active site (POLA1), which is thought to be mediated by the C-terminal domain of the primase large subunit (PRIM2-C) to synthesize dNTPs of approximately 20 nucleotides. The aim of this project was to optimize the expression and purification of Pol α/primase. The insect codon optimized POLA1 was C-terminally Strep tagged and transposed into the baculovirus genome. The other subunits of Pol α/primase, POLA2, PRIM1 and PRIM2 were cloned and expressed in E. coli cells. The cell lysates from Sf9 insect cells and E. coli cells were then mixed and purified by immunoaffinity chromatography and size-exclusion chromatography. This helped us achieve a pure Pol α/primase containing all the four subunits with a good total yield. The identity of all the protein bands were verified by mass spectroscopy. Furthermore, the protein demonstrated primer extension activity on multiple primer/template substrates. We also characterized the effect of the human replication protein A (RPA) on the DNA polymerization activity of Pol α/primase.

No description available.

Abstract DNA polymerase ε from HeLa cells has been purified as a heterodimer of a 261 kDa catalytic subunit and a tightly associated smaller polypeptide, the B-subunit. The cDNAs encoding the B-subunits of both human and mouse Pol ε were cloned and shown to encode proteins with a predicted molecular weight of 59 kDa. These subunits are 90 % identical and share 22 % identity with the 80 kDa B-subunit of Saccharomyces cerevisiae Pol ε. The gene for the human Pol ε B-subunit was localized to chromosome 14q21-q22 by fluorescence in situ hybridization. Primary structure analysis of the Pol ε B-subunits demonstrated that they are similar to the B-subunits of Pol α, Pol δ and archaeal DNA polymerases, and comprise a novel protein family of DNA polymerase associated-B-subunits. The family members have 12 conserved motifs distributed in the C-terminal parts, which apparently form crucial structural and functional sites. Secondary structure predictions indicate that the B-subunits share a similar fold, and phylogenetic analysis demonstrated that the B-subunits of Pol α and ε form one subfamily, while the B-subunits of Pol δ and the archaeal proteins form a second subfamily. The corresponding eukaryotic and archaeal catalytic subunits are not related, but all have the characteristics of replicative DNA polymerases. This indicates that the B-subunits of replicative DNA polymerases from archaea to eukaryotes belong to the same protein family and perform similar functions. In S. cerevisiae, Pol ε associates with the checkpoint protein Dpb11. In this study, a human protein, TopBP1, with structural similarity to the budding yeast Dpb11, fission yeast Cut5 and the breast cancer susceptibility gene product Brca1 was identified. The human TOPBP1 gene localizes to chromosome 3q21-q23 and encodes a phosphoprotein of 180 kDa. TopBP1 has eight BRCT domains and is also closely related to the recently identified Drosophila melanogaster Mus101. TopBP1 expression is induced at the G1/S boundary and it performs an important role in DNA replication, as evidenced by inhibition of DNA synthesis by TopBP1 antiserum in isolated nuclei. TopBP1 also associates with Pol ε and localizes, together with Brca1 to distinct foci in S-phase, but not to sites of ongoing DNA replication. Inhibition of DNA replication leads to re-localization of TopBP1 and Brca1 to stalled replication forks. DNA damage induces formation of distinct TopBP1 foci that co-localize with Brca1 in S-phase, but not in G1-phase. The role of TopBP1 in the DNA damage response is also supported by the interaction between TopBP1 and the human checkpoint protein hRad9. These results implicate TopBP1 in replication and checkpoint functions.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.

DNA polymerase η (pol η), a crucial component of the cellular translesion synthesis pathway, allows cells to bypass and thereby temporarily tolerate DNA damage. Inherited deficiency of pol η, as reported in the variant form of xeroderma pigmentosum, predisposes to UV light-induced skin cancers. To date, pol η is the only DNA polymerase shown to exhibit a causal link to the formation of cancers in humans. However, the role of pol η in the cellular response to forms of DNA damage other than UV-induced lesions is largely unknown. In the first part of this thesis, it is shown that cells deficient in pol η are resistant to ionising radiation. Deficiency in the polymerase was associated with accumulation of cells in S phase of the cell cycle. Cells deficient in pol η demonstrated increased homologous recombination-directed repair of DNA double-strand breaks created by ionising radiation, and depletion of the homologous recombination protein X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells compared to pol η-complemented cells. These findings suggest that homologous recombination mediates S phase-dependent radioresistance associated with pol η-deficiency. In the second part of this thesis, it is shown that pol η-deficient cells have increased sensitivity to the chemotherapeutic compound, oxaliplatin, compared to pol η-deficient expressing cells, but not to the drug 5-fluorouracil that is usually administered in combination with oxaliplatin in the clinical setting. Despite the importance of pol η for cellular survival following exposure to oxaliplatin, the drug did not upregulate the enzyme after either short-term or long-term exposure. Inhibition of pol η activity by siRNA-mediated knockdown of the protein sensitised cells to oxaliplatin treatment, and partially reversed acquired resistance in oxaliplatin-resistant tumour cell lines. These data suggest that pol η is an interesting target whose function can potentially be interfered with to optimise oxaliplatin-based chemotherapy. In the third part of this thesis, clinical samples obtained from oesophageal cancer patients before and after treatment with oxaliplatin-containing chemotherapy were analysed for POLH mRNA levels encoding pol η protein. Malignant tissue specimens obtained before treatment demonstrated a significantly higher level of POLH mRNA than matched normal oesophageal tissue samples. Contrary to the preclinical data, high POLH mRNA expression before therapy was shown to correlate with increased overall and disease-free survival of the patient cohort in the clinical trial. Additionally, patients with high POLH mRNA-expressing cancers had better therapeutic responses (measured by PET-CT) to oxaliplatin-based treatment than those with low levels. These data suggest that POLH mRNA expression should be tested as a biomarker to predict survival and therapeutic responses in oesophageal cancer patients treated with oxaliplatin-containing chemotherapy.

The effectiveness of radiotherapy treatment could be significantly improved if tumour cells could be rendered more sensitive to ionising radiation without altering the sensitivity of normal tissues. However, many of the key mechanisms that determine intrinsic tumour radiosensitivity are largely unknown. This thesis is concerned with the identification of novel determinants of tumour radiosensitivity. A siRNA screen of 200 genes involved in DNA damage repair was conducted using γH2AX foci post-irradiation as a marker of cell damage. This screen identified POLQ as a potential tumour-specific contributor to radioresistance. Subsequent investigations demonstrated that POLQ knockdown resulted in radiosensitisation of a panel of tumour cell lines, whilst having little or no effect on normal tissue cell lines. It was subsequently shown that POLQ depletion rendered tumour cells significantly more sensitive to several classes of cytotoxic agents. Following exposure to etoposide, it was found that tumour cells depleted of POLQ had reduced RAD51 foci formation, suggesting that POLQ is involved in homologous recombination. A homologous recombination assay was used to confirm that POLQ depletion does indeed result in reduced homologous recombination efficiency. These findings led to the investigation of the clinical significance of tumour overexpression of POLQ. The clinical outcomes of patients with early breast cancer were correlated with tumour expression levels of POLQ. It was found that POLQ overexpression was correlated with ER negative disease and high tumour grade, both of which are associated with poor clinical outcomes. POLQ overexpression was associated with extremely poor relapse free survival rates, independently of any other clinical or pathological feature. The mechanism that causes this adverse outcome may in part arise from resistance to adjuvant chemotherapy and radiotherapy treatment. These findings, combined with the limited normal tissue expression of POLQ, make it an appealing target for possible clinical exploitation.

by Au Lai Yin, Cathy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 50-66). / Chapter I. --- ABSTRACT --- p.i / Chapter II. --- ACKNOWLEDGMENTS --- p.iii / Chapter III. --- TABLE OF CONTENTS --- p.iv / Chapter IV. --- LIST OF TABLES --- p.viii / Chapter V. --- LIST OF FIGURES --- p.x / Chapter VI. --- INTRODUCTION --- p.1 / Chapter VII. --- LITERATURE REVIEW --- p.3 / Chapter A. --- Mycobacterial tuberculosis Infections --- p.3 / Chapter B. --- Diagnostic Criteria forM .tuberculosis Infections --- p.3 / Chapter C. --- Mycobacteriological Laboratory Investigations for M. tuberculosis --- p.4 / Chapter 1. --- Conventional methods --- p.4 / Chapter 2. --- Rapid methods --- p.4 / Chapter D. --- Polymerase chain reaction (PCR) - the Principle --- p.5 / Chapter E. --- Application of PCR for Detection of M. tuberculosis --- p.6 / Chapter 1. --- Choice of target sequences --- p.6 / Chapter 2. --- Choice of method for the detection & identification of PCR-amplified product --- p.7 / Chapter 3. --- Studies on pure cultures --- p.9 / Chapter a. --- Detection limit - target DNA --- p.9 / Chapter b. --- Detection limit - Colony forming units --- p.9 / Chapter c. --- Detection limit - Number of cells --- p.10 / Chapter 4. --- Studies on clinical specimens --- p.10 / Chapter 5. --- Problems --- p.12 / Chapter a. --- Availability of target DNA --- p.13 / Chapter (i) --- Cell breakage efficiency --- p.13 / Chapter (ii) --- Target sequence --- p.14 / Chapter b. --- Inhibitory factors for Taq polymerase --- p.14 / Chapter c. --- Contamination --- p.15 / Chapter VIII. --- MATERIALS AND METHODS --- p.16 / Chapter A. --- Bacterial Strains and Strain Maintenance --- p.16 / Chapter 1. --- Reference Strains --- p.16 / Chapter 2. --- Clinical isolates --- p.16 / Chapter B. --- Growth media and culture conditions --- p.17 / Chapter C. --- Restriction Fragment Length Polymorphism (RFLP) --- p.17 / Chapter 1. --- Extraction of chromosomal DNA from M. tuberculosis --- p.18 / Chapter 2. --- Digestion of chromosomal DNA by PVU II --- p.19 / Chapter 3. --- Separation of digested DNA fragment by electrophoresis --- p.19 / Chapter 4. --- Southern Blotting --- p.19 / Chapter 5. --- Preparation of DNA probes by Polymerase Chain Reaction --- p.20 / Chapter 6. --- Hybridization --- p.21 / Chapter 7. --- Detection --- p.21 / Chapter D. --- Assessment of number of organisms --- p.22 / Chapter 1. --- Viable cell count --- p.22 / Chapter 2. --- Direct cell count --- p.22 / Chapter E. --- Assessment of the presence of IS6110/986 in M. tuberculosis isolates --- p.23 / Chapter F. --- Human leukaemic monocytic cell line (THP-1) --- p.23 / Chapter 1. --- Growth media and maintenance --- p.23 / Chapter 2. --- Culture Conditions --- p.24 / Chapter 3. --- Uptake of M. tuberculosis --- p.24 / Chapter G. --- Cell breakage and DNA extraction methodologies --- p.25 / Chapter H. --- Polymerase chain reaction (PCR) methodologies --- p.28 / Chapter 1. --- Primer and probe --- p.28 / Chapter 2. --- PCR conditions --- p.28 / Chapter 3. --- Detection --- p.29 / Chapter I. --- Patients and Clinical specimens --- p.30 / Chapter 1. --- Patients recruitment --- p.30 / Chapter 2. --- Clinical specimens --- p.30 / Chapter IX. --- RESULTS --- p.32 / Chapter A. --- "Development or Selection of a ""Standardized"" PCR Protocol for the Detection of M. tuberculosis Using Pure Cultures In Vitro" --- p.32 / Chapter 1. --- Selection of organisms for verification of the PCR protocol --- p.32 / Chapter 2. --- Optimization of the PCR conditions --- p.32 / Chapter 3. --- Detection limit of target DNA using the PCR procedure --- p.33 / Chapter B. --- Initial Screening of Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 &22a Based on Detection Limits of Colony Forming Units and Number of Cells --- p.34 / Chapter C. --- Comparison of Method 1 and Method 2 Based on Detection Limits of Colony Forming Units and Number of Cells Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis with variable copies of IS6110/986 --- p.34 / Chapter D. --- Detection of M. tuberculosis Isolates Within Macrophages --- p.35 / Chapter 1. --- Uptake of M. tuberculosis cells by THP-1 --- p.35 / Chapter 2. --- Comparison of the Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 & 22a Phagocytized by Activated THP-1 Macrophages --- p.35 / Chapter 3. --- Comparison of Method 1 and Method 2 Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis Phagocytized by Activated THP-1 Macrophages --- p.36 / Chapter E. --- Analysis of Clinical Specimens Using Method 1 & 2 with the Optimized PCR Protocol --- p.36 / Chapter 1. --- Bronchial Aspirate & Bronchoaveolar Lavage Fluid --- p.36 / Chapter 2. --- Pleural Fluid --- p.37 / Chapter 3. --- Tissue --- p.37 / Chapter 4. --- Sputum --- p.38 / Chapter 5. --- Cerebrospinal Fluid --- p.38 / Chapter X. --- DISCUSSION --- p.39 / Chapter A. --- Selection of IS6110/986 for DNA amplification --- p.39 / Chapter B. --- Optimization of PCR conditions reflected by detection limit of target DNA --- p.40 / Chapter C. --- Selection of cell breakage methods based on detection limits of CFU and/or number of mycobacterial cells --- p.41 / Chapter D. --- Application of Methods 1 & 2 and the optimized PCR protocol for clinical specimens --- p.43 / Chapter 1. --- Bronchial aspirates and bronchoaveolar lavage fluids --- p.43 / Chapter 2. --- Pleural fluids --- p.44 / Chapter 3. --- Tissues --- p.45 / Chapter 4. --- Sputa --- p.46 / Chapter 5. --- Cerebrospinal fluids --- p.46 / Chapter XI. --- CONCLUSION --- p.48 / Chapter XII. --- LITERATURE CITED --- p.50 / Chapter XIII --- TABLES --- p.67 / Chapter XIV. --- FIGURES --- p.85

The thermophilic enzyme, Thermus aquaticus (Taq) DNA polymerase, is an essential tool in molecular biology because of its ability to synthesis DNA in vitro and its inherent thermal stability. Taq DNA polymerase is widely used in the polymerase chain reaction (PCR), an essential technique in a broad range of different fields from academic research to clinical diagnostics. The use of PCR-based tests in diagnostic testing is ever increasing; however, many of the samples being tested contain substances that inhibit PCR and prevent target amplification. Many attempts have been made to engineer polymerases not only to increase resistance to overcome the problem of inhibition, but also to enhance other characteristics such as fidelity, processivity and thermostability. Heparin, found in blood samples, and phytate, found in faecal samples, are two examples from a number of known PCR inhibitors. The mode of action of most PCR inhibitors is not well understood, but inhibition is thought to occur by enzyme binding or through the chelation of Mg2+ ions essential for PCR. In this project, a system of directed evolution by compartmentalised self-replication (CSR) was established and successfully employed to screen a mutant library for Taq DNA polymerase variants with enhanced resistance to the inhibitors heparin and phytate. CSR is a recently-established high-throughput method for the creation of novel polymerases, based on a feedback loop whereby polymerase variants replicate their own encoding gene. A mutant library of 106 variants was produced by random mutagenesis error-prone PCR, in which only the polymerase domain of Taq was mutagenised. Firstly, the CSR system was established and tested by performing a screen in the presence of heparin to select for heparin-resistant variants. Characterisation of selected variants revealed that a single round of CSR had produced a Taq variant (P550S, T588S) with a 4-fold increase in heparin resistance. The IC50 was increased from 0.012U/ml heparin to 0.050U/ml heparin. The study with heparin was followed by a phytate screen, in which two rounds of CSR were performed with an initial round of error-prone PCR followed by re-diversification (recombination) of the mutant library using the staggered extension process (StEP). The two rounds of CSR yielded a Taq variant with a 2-fold increase in phytate-resistance compared to the wild-type, with IC50 increased from 360μM phytate to 700μM phytate. The best phytate mutant (P685S, M761V, A814T) was further characterised and it was found that the catalytic activity, thermostability and fidelity of the mutant were comparable to the wildtype enzyme. The position of resistance-conferring mutations of the novel Taq variants evolved in this study provided some evidence for the inhibitors’ predicted modes of action in the case 2 of both phytate and heparin. As phytate’s mode of action is poorly understood, further investigations were performed to elucidate its role in PCR inhibition. A thorough investigation into the importance of relative phytate and Mg2+ levels on PCR was conducted and revealed for the first time convincing evidence that the primary mode of phytatemediated PCR inhibition is by chelation. Further work led to the successful crystallisation of Taq in the presence of phytate, although subsequent X-ray diffraction data to 2.5Å did not reveal phytate bound within the enzyme structure. Site-directed mutagenesis studies were used to probe cross-over between heparin and phytate-conferring mutations. Thus, in addition to providing valuable information for novel Taq variants with a potential application in fecal-based PCR diagnostic tests, this project has begun to provide insight into the fundamental aspects of the mode of action of phytate as a polymerase and PCR inhibitor.

acase@tulane.edu