Nucleases are integral to all DNA processing pathways. The exact nature of substrate recognition and enzymatic specificity in structure-specific nucleases that are involved in DNA replication, repair and recombination has been under intensive debate. The nucleases that rely on the contours of their substrates, such as 5’ nucleases, hold a distinctive place in this debate. How this seemingly blind recognition takes place with immense discrimination is a thought-provoking question. Pertinent to this question is the observation that even minor variations in the substrate provoke extreme catalytic variance. Increasing structural evidence from 5’ nucleases and other structure-specific nuclease families suggest a common theme of substrate recognition involving distortion of the substrate to orient it for catalysis and protein ordering to assemble active sites. Using three single-molecule (sm)FRET approaches of temporal resolution from milliseconds to sub-milliseconds, along with various supporting techniques, I decoded a highly sophisticated mechanism that show how DNA bending and protein ordering control the catalytic selectivity in the prototypic system human Flap Endonuclease 1 (FEN1). Our results are consistent with a mutual induced-fit mechanism, with the protein bending the DNA and the DNA inducing a protein-conformational change, as opposed to functional or conformational selection mechanism. Furthermore, we show that FEN1 incision on the cognate substrate occurs with high efficiency and without missed opportunity. However, when FEN1 encounters substrates that vary in their physical attributes to the cognate substrate, cleavage happens after multiple trials During the course of my work on FEN1, I found a novel photophysical phenomena of protein-induced fluorescence quenching (PIFQ) of cyanine dyes, which is the opposite phenomenon of the well-known protein-induced fluorescence enhancement (PIFE). Our observation and characterization of PIFQ led us to further investigate the general mechanism of fluorescence modulation and how the initial fluorescence state of the DNA-dye complex plays a fundamental role in setting up the stage for the subsequent modulation by protein binding. Within this paradigm, we propose that enhancement and quenching of fluorescence upon protein binding are simply two different faces of the same process. Our observations and correlations eliminate the current inconvenient arbitrary nature of fluorescence modulation experimental design.

abstract: Conformational changes in biomolecules often take place on longer timescales than are easily accessible with unbiased molecular dynamics simulations, necessitating the use of enhanced sampling techniques, such as adaptive umbrella sampling. In this technique, the conformational free energy is calculated in terms of a designated set of reaction coordinates. At the same time, estimates of this free energy are subtracted from the potential energy in order to remove free energy barriers and cause conformational changes to take place more rapidly. This dissertation presents applications of adaptive umbrella sampling to a variety of biomolecular systems. The first study investigated the effects of glycosylation in GalNAc2-MM1, an analog of glycosylated macrophage activating factor. It was found that glycosylation destabilizes the protein by increasing the solvent exposure of hydrophobic residues. The second study examined the role of bound calcium ions in promoting the isomerization of a cis peptide bond in the collagen-binding domain of Clostridium histolyticum collagenase. This study determined that the bound calcium ions reduced the barrier to the isomerization of this peptide bond as well as stabilizing the cis conformation thermodynamically, and identified some of the reasons for this. The third study represents the application of GAMUS (Gaussian mixture adaptive umbrella sampling) to on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of DNA, and made predictions concerning the affinity of Cy3 for different base pairs, which were subsequently verified experimentally. Finally, the adaptive umbrella sampling method is extended to make use of the roll angle between adjacent base pairs as a reaction coordinate in order to examine the bending both of free DNA and of DNA bound to the archaeal protein Sac7d. It is found that when DNA bends significantly, cations from the surrounding solution congregate on the concave side, which increases the flexibility of the DNA by screening the repulsion between phosphate backbones. The flexibility of DNA on short length scales is compared to the worm-like chain model, and the contribution of cooperativity in DNA bending to protein-DNA binding is assessed. / Dissertation/Thesis / Ph.D. Chemistry 2011

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Eurycoma Longfolia Jack, also known as Tongkat Ali (TA) is a tropical plant belonging to the family of Simaroubaceae is widely distributed in South East Asian countries. The extracts of TA have been proven to have cytotoxicity, anti-proliferative and aphrodisiac properties. In vitro assays revealed cytotoxicity toward human breast cancer cell lines MCF-7, KB, CaOV-3, RD, DU-145and HepG2 human liver cancer cells and appear promising as a new chemotherapeutic agent against human cervical carcinoma (HeLa) cells. Although, there are extensive studies reported on its cytotoxicity benefits there are none pertaining to LNCaP human prostate cell line. Therefore, this study aimed at testing the effects of TA on LNCaP cells and prostate specific antigen (PSA) production. Materials and Methods This study investigated the effect of different concentrations of TA (0.0025, 0.025, 2.5, 25 and 250 g/ml) TA on LNCaP human prostate cancer cell line for 24 and 96 hours. The following parameters were investigated: morphology, cell viability (MTT), testosterone modulation, Annexin V-CY3 binding (Apoptosis), DNA fragmentation (TUNEL), caspase 3/7 activity (apoptosis), and PSA production. Results When observing the morphological changes of LNCaP cells exposed to TA, a clear increase in detachment and cell death via apoptosis as the concentrations of TA increased. The viability decreased significantly in both 24 and 96 hour treatment of TA at higher dosages (25 and 250 g/ml). The significant inhibitory effects on testosterone stimulated cell proliferation were seen at TA concentrations as low as 0.0025 μg/ml TA. At higher concentrations of TA (25 and 250 μg/ml), for all testosterone dosages a decreasing trend in proliferation was found. vii Testosterone concentrations of 10 nM showed maximum stimulation of cell proliferation for TA dosages up to 2.5 μg/ml. All concentrations of TA showed significant increase in apoptosis of the cells as dosages increased. A higher amount of DNA damage found at the highest dosage (250 μg/ml) of TA. The relative caspase 3/7 activation showed significant (P=0.0043) activation at the highest concentration (250 μg/ml) of TA. Relative PSA production resulted only a 5% increase with no significant difference at all doses indicting that TA does not change the cell PSA production and the decline in PSA concentration is due to LNCaP cells dying as a result of this exposure to TA. Conclusion In summary, the major finding of this study is that Tongkat Ali inhibits the viability of prostate cancer cell lines (LNCaP) through caspase-mediated pathway, as well as increased the level of apoptotic such as DNA fragmentation. In addition, Tongkat Alin also inhibited PSA production. In LNCaP cells, testosterone with the addition of TA does not increase the growth of the cells. However, more in-vitro and in-vivo studies are needed to establish the exact constituents of the extracts and their mechanism of action. Thus, this study opens perspectives on the use of Tongkat Ali preparations in the treatment of aging male symptoms, prostate cancer prevention or as additional treatment to standard prostate cancer therapy.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / This study investigated the effects of TKIs on the growth and proliferation of MCF-7 breast carcinoma cells in culture. MCF-7 cells were exposed to different concentrations of TKIs alone and in combination with each other. Inhibition of cell growth by TKIs used individually occurred in a dose- and time-dependent manner. When EGFR Inhibitor I, EGFR Inhibitor II/BIBX1382 and the multi-specific EGFR/ErbB-2/ErB-4 Inhibitor were used in combination with each other at equimolar log dose concentrations, the combined effects on cell growth was significantly different to inhibitors used individually as reflected in a decreased EC50 (IC50) during combination treatments. Generally, for the combinations with DOX, CPL and the TKIs, synergistic as well as antagonistic effects were observed at isoeffective concentrations with resultant decreases in dose reduction indices (DRIs) implying greater efficacies with the respective combinations. In this study, conventional PCR was used to detect and illustrate the presence of the EGFR gene in the samples, while RT-qPCR was used to determine the mRNA expression levels of this gene in MCF-7 breast carcinoma cells