This thesis provides an overview of DNA replication proteins that potentially may serve as targets for antimicrobials in drug-resistant pathogens and includes the in silico identification of the... Show moreThis thesis provides an overview of DNA replication proteins that potentially may serve as targets for antimicrobials in drug-resistant pathogens and includes the in silico identification of the replication proteins of C. difficile. Additionally, the mode of action and the current developmental status of the compounds directed at the core replication machinery and accessory replication proteins are discussed (Chapter 2). In chapter 3 we determined the complete genome sequence of our reference laboratory strain, C. difficile 630Δerm, and compared this sequence to the sequence of C. difficile 630. This revealed an unexpected transposition of the mobile genetic element CTn5. The genome sequence now offers an appropriate reference for all molecular work on this strain. Apart from the identification and characterization of gyrase and DNA polymerase in previous studies, no information on DNA replication proteins or the mechanism of replication was available. To address this hiatus, we sought out to identify core replication proteins and to experimentally validate the functional role of these proteins. The findings of this experimental work, with a focus on helicase and primase, are presented in chapter 4 and chapter 5, respectively. Show less
This thesis discusses the purification and activities of the SARS-coronavirus (SARS-CoV) RNA-dependent RNA polymerases (RdRps) nsp12 and nsp(7+8). The first is a large monomeric RdRp, whose... Show moreThis thesis discusses the purification and activities of the SARS-coronavirus (SARS-CoV) RNA-dependent RNA polymerases (RdRps) nsp12 and nsp(7+8). The first is a large monomeric RdRp, whose stability is greatly influenced by N-terminal additions. In contrast, the latter RdRp is a remarkable hexadecameric complex that is capable of both de novo initiation and primer-extension. In addition these RNA polymerase activities, the thesis also expatiates upon the calibration of magnetic tweezers for force measurements and the dynamics of the equine arteritis virus (EAV) RNA helicase as function of NTP hydrolysis. Show less
