Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...

DNA polymerase binds to the double stranded DNA and extends the primer strand by adding deoxyribonucletide to the 3’-end. Several reactions in the polymerase active site have been reported by Kornberg in addition to the polymerization. We observed DNA polymerase I can act as a pyrophosphatase and hydrolyze deoxyribonucletide. In performing the pyrophosphatase activity, DNA polymerase I requires to interact with RNA. RNA in general, was found to activate the DNA polymerase I as pyrophosphatase. This hydrolysis causes depletion of dNTP and inhibits DNA polymeration synthesis in vitro. In this RNA-dependent catalysis, DNA polymerase I catalyzes only dNTP but not rNTP. We have also observed that many other DNA polymerases have this type of the RNA-dependent pyrophosphatase activity. Our experimental data suggest that the exonuclease active sites most likely play the critical role in this RNA-dependent dNTP hydrolysis, which might have a broader impact on biological systems. On the basis of the crystal structure of a ternary complex of RNase H (Bacillus halodurans), DNA, and RNA, we have introduced the selenium modification at the 6-position of guanine (G) by replacing the oxygen (SeG). The SeG has been incorporated into DNA (6 nt. - 6 nucleotides) by solid phase synthesis. The crystal structure and biochemical studies with the modified SeG-DNA indicate that the SeDNA can base-pair with the RNA substrate and serve as a template for the RNA hydrolysis. In the crystal structure, it has been observed that the selenium introduction causes shifting (or unwinding) of the G-C base pair by 0.3 Å. Furthermore, the Se-modification can significately enhance the phosphate backbone cleavage (over 1000 fold) of the RNA substrate, although the modifications are remotely located on the DNA bases. This enhancement in the catalytic step is probably attributed to the unwinding of the local duplex, which shifts scissile phosphate bond towards the enzyme active site. Our structural, kinetic and thermodynamic investigations suggest a novel mechanism of RNase H catalysis, which was revealed by the atom-specific selenium modification.

Mittels NMR-Spektroskopie werden Störungen in Struktur und Dynamik von DNA untersucht, die durch den Einbau jeweils eines der beiden Fluorophore 2- Aminopurin (2AP) und 2-Hydroxy-7-nitrofluoren (HNF) hervorgerufen werden. Zu diesem Zweck werden die NMR-Strukturen der modifizierten Duplexe mit der Sequenz 5’-GCTGCAXACGTCG-3’ berechnet. Im Fall X=2AP (13mer2AP) ist die Partnerbase im Komplementärstrang ein T, während gegenüber X=HNF (13mer- HNF) eine abasische Stelle eingeführt wird. Durch den Vergleich der Ergebnisse zum 13mer2AP mit denjenigen des entsprechenden unmodifizierten DNA Doppelstranges (13merRef, X=A) konnte jegliche Änderung eindeutig dem Einbau von 2AP zugordnet werden. Für die NMR-Strukturen von 13merRef und 13mer2AP können kleine aber signifikante, über die gesamte Helix verteilte Strukturstörungen nachgewiesen werden. Experimente zum Iminoprotonenaustausch mit Wasser ergeben, daß der Einbau von 2AP die Basenpaarlebensdauern der 7 zentralen Basenpaare erniedrigt. Die kürzere Lebensdauer des 2AP:T Basenpaares kann jedoch nicht den schnellen Wasseraustausch im Sättigungstransfer- Experiment ohne Zugabe von Basenkatalysator erklären. Als Erklärung für diese Diskrepanz wird eine effizientere intrinsische Katalyse vermutet. Als mögliche, katalytisch aktive Stelle wird das T O4 Atom diskutiert, welches über die große Furche leicht zugänglich ist und das keine Wasserstoffbrückenbindung innerhalb des Basenpaares ausbilden kann. Die übergeordnete Struktur des 13merHNF ist eine B-Form DNA Helix. Die NOE Kreuzpeaks zu den Protonen im HNF können jedoch nur durch zwei verschiedene Orientierungen des HNFs in der helikalen Anordnung beschrieben werden. Das Verhältnis der beiden Orientierungen untereinander wird als 1:1 abgeschätzt. Störungen in der Basenpaardynamik werden durch die höhere Linienbreite und die starke Hochfeldverschiebung des T auf der 5’-Seite ausgehend von der abasischen Stelle angedeutet. / Structural and dynamic perturbations in DNA upon incorporation of either fluorophore, 2-Aminopurine (2AP) or 2-Hydroxy-7-nitrofluorene (HNF), are characterized by NMR spectroscopy. For this purpose the NMR solution structures of the modified DNA duplexes with the sequence 5’-GCTGCAXACGTCG-3’ are solved. For X=2AP (13mer2AP) the partner base in the complementary strand is T, while for X=HNF (13merHNF) an abasic site is introduced to avoid steric strain. By comparing results on 13mer2AP with the corresponding unmodified DNA duplex (13merRef, X=A), any perturbation can be unambiguously assigned to 2AP incorporation. For the NMR solution structure of 13merRef and 13mer2AP small but significant changes in helical parameters are found throughout the helix. Imino proton exchange measurements reveal an extended, distributed effect of 2AP incorporation on the lifetimes of the central seven base pair. However, the reduced base pair lifetime of 2AP:T cannot fully account for the rapid water exchange observed with saturation transfer experiments in the absence of base catalyst. This indicates enhanced intrinsic catalysis. As a possible catalytic site the T O4 atom opposite 2AP is discussed, which is easily accessible through the major groove and lacks a hydrogen bonding partner within the base pair. The overall NMR solution structure is found to be B-DNA. However the NOE cross-peaks involving the HNF residue can only be accounted for by two different orientations of the HNF inside the DNA helical stack. Their population ratio is estimated to be 1:1. Dynamical perturbation is indicated by the increased linewidth and strong upfield shift of the T residue to the 5’-side of the abasic site.

A significant focus of nucleic acids research is on the reactivity of electrophilic species with DNA to form addition products (adducts). Phenols are known to be able to form adducts at the C8 site of deoxyguanosine (dG). This dissertation studies the oxygen (O)-linked class of phenolic dG adducts for their hydrolytic stability as well as their structural impact on the DNA duplex. To determine the effect of C8 O-linked phenolic dG adducts on glycosidic bond stability spectrophotometric determination of hydrolysis kinetics was performed. The kinetics establish the adducts to be less stable than native dG in acid, but surprisingly stable under physiological conditions. Then to assess the modified duplex structure, a C8 O-linked phenolic dG adduct was incorporated into a DNA duplex. Thermal melting analysis establish the adduct as having a destabilizing effect on the regularly paired duplex and the conformational analysis suggests the phenolic lesion to be weakly mutagenic. / NSERC