Cells have lesion bypass DNA polymerases (DNAPs), often in the Y-Family, which synthesize passed DNA damage. One class of Y-Family DNAPs includes hDNAP k, EcDNAP IV and SsDbh, which insert accurately opposite N2-dG adducts, including BP-N2-dG formed from benzo[a]pyrene (BP). Another class includes hDNAP h, EcDNAP V and SsDpo4, which insert accurately opposite UV-damage. For correct Watson-Crick pairing between BP-N2-dG and dCTP, the BP moiety must be in the minor groove. On the minor groove side of the active site, k/IV/Dbh-class DNAPs have large openings that accommodate the BP moiety. Primer extension assays with purified proteins show that DNAP IV correctly inserts dCTP opposite BP more than 10-fold faster than it mis-inserts dATP, dGTP, or dTTP. In contrast, h/V/Dpo4-class DNAPs have small active site openings, which cannot accommodate BP and lead to a distorted structure and increased mutagenesis; e.g., Dpo4 has dGTP and dATP insertion rates that are 10-fold greater than those of dCTP. The opening in Dpo4 is plugged and bulky, whereas DNAP IV has a relatively spacious cavity. Consistent with this model, mutants of Dpo4 with a larger opening insert up to 10-fold more accurately opposite BP-N2-dG. Near the active site, Dpo4 has a single non-covalent bridge (NCB) between the little finger domain and the thumb-palm-fingers domain. DNAP IV and Dbh have a second, distal NCB that is 8 angstroms away from the active site towards the 3' end of the template DNA. Dpo4 becomes nearly 5-fold more accurate when mutated to carry a distal NCB, suggesting that NCB's also help control mutagenesis. Lastly, the active site of Dpo4 has a cavity in the major groove side, which may allow base flipping and dGTP insertion opposite -BP, while k/IV/Dbh-type polymerases do not. When this cavity is plugged in Dpo4 by mutagenesis or the introduction of an N-clasp motif, dGTP rates increase by nearly 20-fold. In conclusion, this data suggests that three structural regions contribute to accurate dCTP insertion opposite BP-N2-dG by k/IV/Dbh-class DNAPs: a large opening on the minor groove side near the active site, a cavity on the major groove side, and the number of non-covalent bridges between the little finger domain and the thumb-palm-fingers domain.

Published information on the structure and regulation of H. pylori promoters is limited. The work presented in this thesis describes the cloning and characterisation of promoter regions from a clinical isolate of H. pylori, and the development of an alternative, non-radioactive method for verifying the location of transcriptional start sites of bacterial promoters. H. pylori 4187E promoters were randomly cloned into the promoter-trap vector pKK232-8 in Escherichia coli DH5α using two sets of restriction enzymes. Vector pKK232-8 contains a promoterless chloramphenicol acetyltransferase (CAT) gene. Seventy-four promoter-containing clones were isolated from selective media based on their resistance to chloramphenicol. The strength of each promoter was analysed qualitatively, using chloramphenicol minimum inhibitory concentrations, and quantitatively, using CAT assays following exposure of the clones to pH 4 and pH 7. Selected promoter fragments were subcloned into the GFP reporter vector pFPV25, containing a promoterless gfp gene. The subclones were exposed to buffered LB broth at pH 4, 5, 6, 7 and 8, for varying lengths of time, to study acid-induced regulation of gene expression. Subclones were examined qualitatively, using visual examination of GFP fluorescence and fluorescence microscopy, and quantitatively, using flow cytometry following acid shock. DNA sequences were determined for 61 of the 74 H. pylori promoters, and sequence alignments with the published H. pylori strains (26695 and J99) were performed. The transcriptional start site of 27 H. pylori promoter fragments was experimentally mapped using a fluorescence-based primer extension protocol developed by our group. Potential -35 and -10 sequences were identified for each promoter, and a new consensus sequence for H. pylori promoters was proposed based upon these results. This study has considerably expanded knowledge of H. pylori promoter sequences and transcriptional start sites based on those which also function in E. coli. It has also revealed several H. pylori promoters which appear to respond to acid stress

All living organisms are made of cells and they store their hereditary information in the form of double stranded DNA. In all organisms DNA replication and repair is essential for cell division and cell survival. These processes require deoxyribonucleotides (dNTPs), the building blocks of DNA. Ribonucleotide reductase (RNR) is catalyzing the rate limiting step in the de novo synthesis of dNTPs. Active RNR is a heterodimeric protein complex. In S phase cells, the mouse RNR consists of the R1 and the R2 proteins. The R1/R2 RNR-complex supplies the cell with dNTPs required for DNA replication. Outside S-phase or in non-proliferating cells RNR is composed of R1 and p53R2 proteins. The R1/p53R2 RNR-complex supplies cells with dNTPs required for mitochondrial DNA replication and for DNA repair. An undisturbed dNTP regulation is important since unbalanced dNTP pools results in DNA mutations and cell death. Since unbalanced pools are harmful to the cell, RNR activity is regulated at many levels. The aim of this thesis is to study how the mouse RNR genes are regulated at a transcriptional level. We have focused on the promoter regions of all three mouse RNR genes. Primer extension experiments show that the transcription start of the TATA-less p53R2 promoter colocalizes with an earlier unidentified initiator element (Inr-element). This element is similar to the known Inr-element in the mouse R1 promoter. Furthermore, functional studies of the R1 promoter revealed a putative E2F binding element. This result suggests that the S phase specific transcription of the R1 gene is regulated by a similar mechanism as the R2 promoter which contains an E2F binding site. Finally we have established a method to partially purify the transcription factor(s) binding the upstream activating region in the mouse R2 promoter by phosphocellulose chromatography and affinity purification using oligonucleotides immobilized on magnetic beads. This method will allow us to further study the transcription factors responsible for activating expression of the R2 protein. This method has a potential to be utilized as a general method when purifying unknown transcription factors.