With respect to a multienzyme complex of deoxyribonucleoside triphosphate (dNTP) synthesis somehow juxtaposed with DNA replication sites, our laboratory has demonstrated the existence of a multienzyme complex in T4-infected E. coli, named the T4 dNTP synthetase complex, but the idea of direct linkage of dNTP synthesis to DNA replication and organization of the complex has not been well established. This study had two objectives. The first objective was to test the specific hypothesis that gp32, the single-stranded DNA binding protein encoded by gene 32, plays a role in recruiting enzymes of dNTP synthesis to the replisome and in organizing the dNTP synthetase complex. By use of two newly created gene 32 mutants along with several experimental approaches, DNA-cellulose chromatography, coimmunoprecipitation, and glutathione-S-transferase pull downs, interactions of gp32 with thymidylate synthase (gptd), ribonucleotide reductase (gpnrdA/B), and E. coli NDP kinase have been identified. These results support the hypothesis that gp32 helps to recruit enzymes of dNTP synthesis to DNA replication sites. As the second objective, I investigated contributions of two host proteins, E. coli nueleoside diphosphate kinase (NDP kinase) and adenylate kinase (Adk), to the organization of the complex. As an important step to understand roles of E. coli NDP kinase in the complex, I identified direct interactions of E. coli NDP kinase with gpnrdA/B, dCMP hydroxymethylase (gp42), and dihydrofolate reductase (gpfrd) by means of coimmunoprecipitation and glutathione-S-transferase pull-down experiments. Interestingly, these interactions were influenced by the presence of substrate nucleotides or an analog for E. coli NDP kinase, suggesting that metabolite flux may affect the preference of E. coli NDP kinase binding to enzymes in the complex in vivo. Meanwhile, Adk involvement in DNA precursor synthesis has been suggested, particularly in phage T4-infected E. coli, from observations of increased thermostability of temperature-sensitive Adk in situ. The involvement of E. coil Adk in the complex was demonstrated by identifying some proteins of the T4 dNTP synthetase complexgp42, dNMP kinase (gpl), gpfrd, and E. coli NDP kinasedirectly interacting with Adk, implying that E. coil Adk would be properly located in the complex to efficiently carry out the conversion of dNDPs to dNTPs. This implication was supported by measurements of T4 DNA synthesis. Taken together, this research strongly supports the idea of connection of dNTP synthesis to DNA replication and allows us to take a step toward understanding the organization of the complex at DNA replication sites. / Graduation date: 2005

Nitrogen is an essential building block of proteins and nucleic acids and, therefore, crucial for the biosphere. Nearly 79 % of the air consists of nitrogen, but in the form of nitrogen gas (N2), which cannot be utilized by most organisms. Nitrogen-fixing microorganisms such as cyanobacteria have a central role in supplying biologically useful nitrogen to the biosphere. Therefore, it is important to achieve further understanding of control mechanisms involved in nitrogen fixation and related processes. This thesis concerns different molecular aspects of the transcription factor NtcA from the heterocystous cyanobacterium Anabaena PCC 7120. Apart from performing oxygenic photosynthesis, Anabaena PCC 7120 is also capable of fixing nitrogen. NtcA is a protein regulating transcription of a wide range of genes and in particular genes involved in cyanobacterial global nitrogen control. NtcA binds as a dimer to the promoter regions of target genes such as those involved in nitrogen fixation and heterocyst differentiation. NtcA from Anabaena PCC 7120 was heterologously expressed in E. coli and a high yield of recombinant protein was achieved through purification by Ni-IMAC chromatography. The purified NtcA was used to examine DNA binding motifs preferred by NtcA in vitro using a semi-random library of DNA sequences. The preferred binding sequence for NtcA is TGTA – N8 – TACA and at least five of the bases in the palindromic binding site are necessary for binding. Differences in the consensus sequence in vivo may reflect variations in the structural conformation of NtcA under various physiological conditions. Since an earlier study suggested redox-regulated NtcA-DNA binding the role of the two cysteine residues of NtcA were investigated. Binding studies using three mutants, Cys157Ala, Cys164Ala, and Cys157Ala / Cys164Ala, demonstrated that all these NtcA variants bind to DNA with a slightly higher affinity in the presence of the reducing agent DTT. The studies indicate that the binding mechanism is not dependent on a conformational change of NtcA caused by breaking of intra-molecular disulfide bonds. Crystallization followed by structural studies rendered a partial crystal structure of NtcA. The structure verifies that NtcA is a dimeric protein. Each subunit has three domains: the N-terminal domain, a dimerization helix connecting the N-terminal domain with the C-terminal domain, as well as making up the dimer interface, and a C-terminal domain including the DNA binding helix-turn-helix motif. Furthermore, an NtcA binding site was found in the promoter region of the hupSL gene, encoding an uptake hydrogenase in Nostoc punctiforme (ATCC 29133), indicating that yet another gene is transcriptionally controlled by NtcA, thereby further emphasizing the multifaceted role of NtcA in cyanobacteria.

<p>Hundreds of different factors adorn the eukaryotic genome, binding to it in large number. These DNA binding factors (DBFs) include nucleosomes, transcription factors (TFs), and other proteins and protein complexes, such as the origin recognition complex (ORC). DBFs compete with one another for binding along the genome, yet many current models of genome binding do not consider different types of DBFs together simultaneously. Additionally, binding is a stochastic process that results in a continuum of binding probabilities at any position along the genome, but many current models tend to consider positions as being either binding sites or not.</p><p>Here, we present a model that allows a multitude of DBFs, each at different concentrations, to compete with one another for binding sites along the genome. The result is an 'occupancy profile', a probabilistic description of the DNA occupancy of each factor at each position. We implement our model efficiently as the software package COMPETE. We demonstrate genome-wide and at specific loci how modeling nucleosome binding alters TF binding, and vice versa, and illustrate how factor concentration influences binding occupancy. Binding cooperativity between nearby TFs arises implicitly via mutual competition with nucleosomes. Our method applies not only to TFs, but also recapitulates known occupancy profiles of a well-studied replication origin with and without ORC binding.</p><p>We then develop a statistical framework for tuning our model concentrations to further improve its predictions. Importantly, this tuning optimizes with respect to actual biological data. We take steps to ensure that our tuned parameters are biologically plausible.</p><p>Finally, we discuss novel extensions and applications of our model, suggesting next steps in its development and deployment.</p> / Dissertation

Diaminopropionate ammonia lyase (DAPAL) which belongs to the  class of PLP enzymes is reported only from prokaryotes. It is involved in the removal of two amino groups from its substrate, diaminopropionate, to form ammonia and pyruvate. DAPAL from Escherichia coli (eDAPAL) and Salmonella typhimurium (sDAPAL) was cloned, over expressed and purified using either affinity chromatography or conventional procedures. It was observed that eDAPAL (90 units / mg) was comparatively less active than sDAPAL (200 units / mg). Also the enzymes with the N-terminal His tag were found to be many fold less active than the enzymes without tag. DAPAL had a characteristic absorption maximum at 414nm due to the Schiff`s linkage between PLP and the € - amino group of the active site lysine residue. The apoenzyme was prepared by reaction with L-cysteine, and the resulting thiazolidine complex was easily dialyzed. On reconstitution with PLP, complete regain of absorption spectrum and 60% activity was seen. All the three enzymes (apo-, holo and reconstituted), when subjected to gel filtration chromatography were found to be homodimers of 88 kDa. The active site lysine 78 was mutated to glutamine, and the enzyme was purified to homogeneity. In the mutant enzyme PLP continued to be bound at the active site, but in a different orientation with an absorbance maximum at 406nm. The K78Q enzyme had negligible activity as compared to the wild type enzyme confirming the role of K78 in catalysis. Only a few of the enzymes of the  class have been investigated for their unfolding pathways. Urea induced unfolding studies on sDAPAL revealed that at lower concentrations of urea there was a loss in activity due to the disruption of Schiff's linkage. No gross conformational changes were observed at these concentrations of urea as seen from fluorescence and gel filtration experiments. Increase in concentration of urea led to unfolding of the protein thereby causing a shift in fluorescence maximum from 340nm to 357 nm due to the exposure of the buried tryptophans to the less hydrophobic environment. A considerable amount of aggregation was seen at intermediate urea concentrations, which was possibly the reason for the inability of the protein to refold completely. Based on the results, a concerted mechanism for dissociation and unfolding was proposed for sDAPAL. Aminooxy compounds, which are mechanism-based inhibitors for PLP enzymes have been used as drugs against various disorders for the last few decades. In order to probe the mechanism and efficiency with which these compounds inhibit sDAPAL, cycloserine (D and L), methoxyamine (MA) and aminooxyacetic acid (AAA) were chosen for the inhibition studies. The inhibition rates were measured by monitoring decrease in absorbance at 414nm, increase in the range of 320-330nm due to the product formation and loss of activity upon incubation with the inhibitor. It was seen that both the enantiomers of cycloserine were equally effective in disrupting the Schiff’s linkage with the second order rate constants of 15.8 and 36 M -1 sec –1 respectively. Spectral measurements showed two isosbestic points in the case of DCS and one in the case of LCS. Product of this inhibition reaction was identified to be a heat and acid stable compound namely a hydroxyisooxazole derivative of PMP. It was similar in nature to that reported from GABA aminotransferase. These results showed that unlike in the case of alanine racemase, sDAPAL could be inhibited equally well by both the enantiomers. The inhibition studies with the other two inhibitors namely AAA and MA, showed AAA to be more efficient at disrupting the Schiff’s linkage and causing inactivation of the enzyme. The visible absorbance spectrum showed a single isosbestic point in both the cases, indicative of a single step involved in the formation of the final product. The elution profile of the product of the enzymatic as well as non-enzymatic reactions on a C-18 HPLC column was similar and the product was identified to be an oxime. These inhibitors reacted with sDAPAL many fold better than the other PLP dependent enzymes and therefore these compounds can serve as potential drugs for sDAPAL.

published_or_final_version / Anaesthesiology / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

The fate of DNA from dead cells is an important issue when interpreting results from root canal infections analysed by the PCR technique. DNA from dead bacterial cells is known to be detectable long time after cell death and its stability is dependent on many different factors. This work investigated factors found in the root canal that could affect the recovery of microbial DNA. In an ex vivo experiment, DNA from non-viable gram-positive Enterococcus faecalis was inoculated in instrumented root canals and recovery of DNA was assessed by PCR over a two-year period. DNA was still recoverable two years after cell death in 21/25 teeth. The fate of DNA from the gram-negative bacteria Fusobacterium nucleatum and the gram-positive Peptostreptococcus anaerobius was assessed in vitro. DNA from dead F. nucleatum and P. anaerobius could be detected by PCR six months post cell death even though it was clear that the DNA was released from the cells due to lost of cell wall integrity during the experimental period. The decomposition rate of extracellular DNA was compared to cell-bound and it was evident that DNA still located inside the bacterium was much less prone to decay than extracellular DNA. Free (extracellular) DNA is very prone to decay in a naked form. Binding to minerals is known to protect DNA from degradation. The fate of extracellular DNA was assessed after binding to ceramic hydroxyapatite and dentine. The data showed that free DNA, bound to these materials, was protected from spontaneous decay and from enzymatic decomposition by nucleases. The main conclusions from this thesis were: i) DNA from dead bacteria can be detected by PCR years after cell death ex vivo and in vitro. ii) Cell-bound DNA is less prone to decomposition than extracellular DNA. iii) DNA is released from the bacterium some time after cell death. iv) Extracellular DNA bound to hydroxyapatite or dentine is protected from spontaneous decomposition and enzymatic degradation.

In mammals, circadian rhythms are generated by transcriptional-translational feedback loops consisting of a set of clock genes and their protein products. Among them, Bmal1 is a critical clock gene in generating and maintaining circadian rhythms. Moreover, orphan nuclear receptors REV-ERBs and RORs were known to respectively repress and activate Bmal1 transcription. In our study, we further demonstrated that: (1) REV-ERBalpha might be the main regulator in maintaining Bmal1 oscillation in thymus. (2) Rorgamma mRNA is constant in muscle and testis, and rhythmic in liver, while Rorgammat mRNA is only expressed in thymus, at constant levels. Moreover, the expressions of these two Rorgamma isoforms are affected in Clock mutant mice in a distinct way. (3) RORgamma and RORgammat can activate Bmal1 transcription at a similar level. (4) Rorgamma is a clock-controlled gene. Altogether, our results suggest that the crucial role of REV-ERBs and RORs in peripheral clocks. Furthermore, our work highlights functional differences among mammalian peripheral clocks, which provides important insights into the complexity of the circadian system.

TAL-1, ectopically expressed in 60% of T-cell acute lymphoblastic leukemia (T-ALL) patients, may contribute to poor chemotherapy response. This research sought to determine if TAL-1 influences expression of proteins involved in the NF-kB signaling pathway and thus, resistance to cell death. NF-kB, IKKy, and TRAF-2 expression levels were found to be TAL-1 dependent. Cell death levels were higher in staurosporine-treated cells compared to tumor necrosis factor a-treated or dual-treated cells. TAL-1, NF-kB, IKKy, and TRAF-2 expression levels were elevated in tumor necrosis factor a-treated cells and reduced in staurosporine-treated or dual treated cells compared to untreated cells. These results suggest TAL-1 influences expression of proteins involved in the NF-kB signaling pathway, thus inducing an anti-apoptotic response in the cell. / Department of Biology