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- Pourbozorgi Langroudi, Parham, 1984-
(författare)
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Insights into the roles of the essential Pfh1 DNA helicase in the nuclear and mitochondrial genomes
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Eukaryotic cells have two sets of genomes, the nuclear and mitochondrial, and both need to be accurately maintained. Also, the rate of transcription must be precisely regulated in these genomes. However, there are many natural barriers that dysregulate these processes. The aim of this thesis was to enhance our understanding of the Schizosaccharomyces pombe, Pif1 family helicase, Pfh1, and its roles in the nuclear and mitochondrial genomes. The S. pombe genome contains 446 predicted Gquadruplex (G4) structures. By circular dichroism and Thioflavin-T assay we demonstrated that sequences from the ribosomal DNA (rDNA) and telomeres form G4 structures in vitro. The recombinant nuclear isoform of Pfh1 bound and unwound these G4 structures. Also, by chromatin immunoprecipitation combined with quantitative PCR (ChIP-qPCR), we showed that Pfh1 binds these sequences in vivo. This work provides evidence that G4 structure formation in the rDNA and telomere regions is biologically important and that unwinding of G4 structures is a conserved property of Pif1 family helicases. Using ChIP-seq we found that Pfh1 binds to natural fork barriers, such as highly transcribed genes, and nucleosome depleted regions, and that replication through these sites were dependent on Pfh1. By immunoaffinity precipitation combined with mass spectrometry, Pfh1 interacted with several replisome components, as well as DNA repair proteins, and mitochondrial proteins. Furthermore, Pfh1 moved with similar kinetics as the leading strand polymerase. These findings suggest that Pfh1 is needed at natural fork barriers to promote fork progression, and that it is not just recruited to its target sites but moves with the replisome. Based on these findings, we anticipated that lack of Pfh1 would affect expression of highly transcribed genes. By performing genome-wide transcriptome analysis of S. pombe in the absence of Pfh1, we showed that highly transcribed genes are downregulated more often than other genes. Furthermore, combining absence of Pfh1 together with Topoisomerase 1 (Top1), resulted in slower cell growth, reduced DNA synthesis rate compared to single mutants, and upregulation of genes associated with DNA repair and apoptosis. These data suggest that, cells lacking both Pfh1 and Top1 have severe problem in maintaining their genomes. By ChIP-qPCR analysis we showed that Pfh1 and Top1 directly bind to mitochondrial DNA. In addition, these cells upregulated many metabolic pathways and lost about 80% of their mtDNA. These data suggest that both Pfh1 and Top1 are required for maintenance of mtDNA. This is the first evidence showing that Top1 is present in S. pombe mitochondria. In conclusion, Pfh1 directly binds mitochondrial DNA, and natural fork barriers in the nuclear DNA, such as G4 structures. In the nucleus, Pfh1 is part of the replisome. Cells lacking Pfh1 and Top1 grow slower, rapidly lose their mitochondrial DNA, have slower nuclear DNA synthesis, and induce apoptotic pathways. Finally, this thesis emphasizes the importance of both Pfh1 and Top1 in maintaining the nuclear and mitochondrial genomes.
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| 2. |
- Berry, Bruce W., 1974-
(författare)
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Using de novo design proteins to explore tyrosine radicals and cation-π interactions
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Redox cofactors and amino-acid free radicals play important roles in biology. Although many of the same cofactors and amino acids that form these radicals are found across a broad range of biological systems, identical cofactors can have different reduction potentials. The local environment plays a role in defining these redox potentials. An understanding of this local-environment effect can shed more light on how redox chemistry works in nature. Our laboratory has developed a library of model proteins that are well suited to study amino-acid radicals. a3X is a de novo designed protein that is composed of 67 residues. It forms a three-helix bundle connected by two glycine loops. The radical site is located at position 32 on the central a-helix. The a3X protein is designed to be well-folded and thermodynamically stable across a broad pH range. Paper 1 describes the structural and electrochemical characterization of a3Y, a tyrosine variant of a3X. We were able to obtain a unique Faradaic response from Y32 at both low and high pH, using differential pulse voltammetry. In addition, we successfully redesigned α3Y by introducing a histidine in close proximity to Y32, creating a tyrosine/histidine pair. Our goal in creating this pair was to study proton-coupled electron transfer (PCET) in a well-structured and solvent-sequestered protein environment. In paper 2 we illustrated the redox reversibility of Y32 and produced the first ever Pourbaix diagram for a tyrosine radical in a protein. The formal potential of the Y32-OŸ/Y32-OH redox couple was determined to be 918 ± 2 mV vs. the normal hydrogen electrode (NHE) at pH 8.40. While at pH 5.52, the formal potential of the Y32-OŸ/Y32-OH redox couple was recorded at 1.07 V. Papers 3 and 4 utilize a3W to study cation-π interactions. In paper 3, we showed how solvation can affect the strength of these interactions by -0.9 kcal/mol. In Paper 4, we were able to monitor the disruption of the cation-π interaction with the use of high-pressure fluorescence and were able to calculate the interaction energy for a solvent exposed cation-π. The aim of the work described in this thesis was to use model proteins to study tyrosine radicals to gain a broader perspective and better understanding of the versatility of biological electron transfer and to measure cation-π interactions and how they behave in different environments.
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| 3. |
- Jonna, Venkateswara Rao, 1980-
(författare)
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Class I Ribonucleotide Reductases : overall activity regulation, oligomerization, and drug targeting
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ribonucleotide reductase (RNR) is a key enzyme in the de novo biosynthesis and homeostatic maintenance of all four DNA building blocks by being able to make deoxyribonucleotides from the corresponding ribonucleotides. It is important for the cell to control the production of a balanced supply of the dNTPs to minimize misincorporations in DNA. Because RNR is the rate-limiting enzyme in DNA synthesis, it is an important target for antimicrobial and antiproliferative molecules. The enzyme RNR has one of the most sophisticated allosteric regulations known in Nature with four allosteric effectors (ATP, dATP, dGTP, and dTTP) and two allosteric sites. One of the sites (s-site) controls the substrate specificity of the enzyme, whereas the other one (a-site) regulates the overall activity. The a-site binds either dATP, which inhibits the enzyme or ATP that activates the enzyme. In eukaryotes, ATP activation is directly through the a-site and in E. coli it is a cross-talk effect between the a and s-sites. It is important to study and get more knowledge about the overall activity regulation of RNR, both because it has an important physiological function, but also because it may provide important clues to the design of antibacterial and antiproliferative drugs, which can target RNR.Previous studies of class I RNRs, the class found in nearly all eukaryotes and many prokaryotes have revealed that the overall activity regulation is dependent on the formation of oligomeric complexes. The class I RNR consists of two subunits, a large α subunit, and a small β subunit. The oligomeric complexes vary between different species with the mammalian and yeast enzymes cycle between structurally different active and inactive α6β2 complexes, and the E. coli enzyme cycles between active α2β2 and inactive α4β4 complexes. Because RNR equilibrates between many different oligomeric forms that are not resolved by most conventional methods, we have used a technique termed gas-phase electrophoretic macromolecule analysis (GEMMA). In the present studies, our focus is on characterizing both prokaryotic and mammalian class I RNRs. In one of our projects, we have studied the class I RNR from Pseudomonas aeruginosa and found that it represents a novel mechanism of overall activity allosteric regulation, which is different from the two known overall activity allosteric regulation found in E. coli and eukaryotic RNRs, respectively. The structural differences between the bacterial and the eukaryote class I RNRs are interesting from a drug developmental viewpoint because they open up the possibility of finding inhibitors that selectively target the pathogens. The biochemical data that we have published in the above project was later supported by crystal structure and solution X-ray scattering data that we published together with Derek T. Logan`s research group.We have also studied the effect of a novel antiproliferative molecule, NSC73735, on the oligomerization of the human RNR large subunit. This collaborative research results showed that the molecule NSC73735 is the first reported non-nucleoside molecule which alters the oligomerization to inhibit human RNR and the molecule disrupts the cell cycle distribution in human leukemia cells.
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| 4. |
- Popović-Bijelić, Ana, 1976-
(författare)
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Activation and inhibition of diiron and iron-manganese ribonucleotide reductases
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ribonucleotide reductase (RNR) catalyses the reduction of ribonucleotides to deoxyribonucleotides. In conventional class I RNRs the active site is located in the R1 subunit, and the R2 subunit contains a diiron cofactor and a stable tyrosyl radical essential for activity. Class Ic Chlamydia trachomatis RNR lacks the tyrosyl radical and uses a Mn(IV)Fe(III) cofactor for catalysis. The requirement for metals for RNR activation was studied in C. trachomatis F127Y and Y129F R2, and in mouse wild type and Y177F R2 proteins. The results indicate that R2 affinity for metals is determined by the amino acid located next to the metal site, at the position of the radical carrying tyrosyl. Specifically, R2 proteins that contain phenylalanine in this place bind Mn and Fe, and the tyrosyl containing R2s bind only Fe. Further results show that C. trachomatis RNR can be inhibited by R2 C-terminal oligopeptides. The highest inhibition was observed for a 20-mer peptide indicating that the oligopeptide inhibition mechanism of class Ic is similar to that of the class Ia and b. The second part of the thesis deals with class Ia RNR inhibition. The results show that a lanthanum complex containing three 1,10-phenantroline molecules (KP772) which has showed promising cytotoxic activity in cancer cell lines inhibits mouse R2 protein in the presence of external reductants by iron-chelation. It is suggested that KP772 has several synergistic inhibition mechanisms that contribute to its overall anticancer activity. Moreover, other results show that Triapine, a promising chemotherapeutic compound, and its Fe, Ga, Zn, and Cu complexes, inhibit mouse R2 in both reducing and non-reducing conditions. Inhibition by Triapine involves the binding of the drug to the surface of the R2 protein leading to labilization of the Fe-center and subsequent Fe-chelation by Triapine. Formation of the Fe(II)-Triapine complex which reacts with O2 to produce reactive oxygen species results in complete RNR inactivation.
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