Browsing by Subject "Mutagenesis"

Human physiology is heavily influenced by the colonization of microbes in the gastrointestinal tract. A major roadblock to understanding this process is our inability to genetically manipulate new bacterial species and experimentally assess the function of their genes. In order to map bacterial genes, we describe an application of chemical mutagenesis followed by population-based genomic sequencing. We chose to map genes responsible for motility in Exiguobacterium acetylicum, a representative intestinal Firmicutes bacterium that is intractable to molecular genetic manipulation. We derived strong associations between mutations in 57 E. acetylicum genes and impaired motility and also discovered new motility genes that were previously uncharacterized. We confirmed the genetic link between individual mutations and loss of motility for several of these genes by performing a large-scale analysis of spontaneous suppressor mutations. Furthermore, we generated isogenic strains that allowed us to establish that Exiguobacterium motility is important for the colonization of its vertebrate host.

This methological advance in gene functional analysis of genetically intractable microbes has enabled us to identify 902 essential genes that are directly responsible for growth and survival. This is achieved by large-scale mutant sequencing analysis. By curating the gene list, we assigned the essentiality of genes to uncharacterized genes as well. These results indicate that the genetic dissection of a complex trait, functional annotation of new genes, and the generation of mutant strains can all be accomplished in bacteria without the development of species-specific molecular genetic tools. Ultimately, this advance helps define the role of genes in complex environments.

To investigate the effect of silent mutations in a gene, we have designed and created thousands of AcGFP codon-variant libraries to determine the relationship between codon usage and protein expression. mRNA structures near the initial start codon regions are prominent factor for determining protein expression level, but variation in sequence beyond the start codon region also importantly modulates expression levels.

Topoisomerase 2 (Top2) is an enzyme that helps maintain genome integrity by resolving topological structures that arise during cellular processes such as replication and transcription. To resolve these structures, a Top2 dimer creates a transient double-strand break (DSB) in the DNA. Each subunit forms a phosphotyrosyl bond with the 5’ ends of the break, and this DNA-protein intermediate is called a Top2 cleavage complex (Top2cc). Following the passage of an intact duplex, Top2 re-ligates the DNA and is released to restore genome integrity. Top2cc stabilization by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. This thesis characterizes two novel top2 mutants, both of which are associated with a mutation signature characterized by de novo duplications. These duplication events are dependent on clean removal of the Top2cc from the DNA and DSB repair by nonhomologous end-joining. The first mutant (top2-FY,RG) was identified through a screen for etoposide hypersensitivity, and it generates a stabilized cleavage intermediate in vitro. The second mutant (top2-K720N) is the yeast equivalent of a somatic mutation in TOP2A identified in gastric cancers and choloangiocarcinomas that is also associated with a duplication mutation signature (ID17). Overall, the findings in this thesis are relevant for clinical use of chemotherapeutic drugs that target Top2 and have implications for genome evolution.

Preserving genome stability is critical to ensure the faithful transmission of intact genetic material through each cell division. One of the key components of this preservation is maintaining low levels of mutagenesis. Most mutations arise during replication of the genome, either as polymerase errors made when copying an undamaged DNA template or during the bypass of DNA lesions. Many different DNA repair proteins act both prior to and during replication to prevent the occurrence of these mutations. Although the mechanisms by which mutations occur and the various repair proteins that act to suppress mutagenesis are conserved throughout all species, they are best characterized in the yeast Saccharomyces cerevisiae. In this work, we have used this model system to study two types of spontaneous mutagenesis: oxidative mutagenesis and frameshift mutagenesis. In the first part of this work, we have examined mutagenesis that arises due to one of the most common oxidative lesions in the cell, 7,8-dihydro-8-oxoguanine or GO. When present during replication, these GO lesions generate characteristic transversion events that are accurately repaired by the mismatch repair pathway. We provide the first evidence that a second pathway involving the translesion synthesis polymerase Pol&eta acts independently of the mismatch repair pathway to suppress GO-associated mutagenesis. We have also examined how differences in replication timing during S phase contribute to variations in the rate of these mutations across the genome. In the second part of this work, we have examined how spontaneous frameshift mutations are generated during replication. While most frameshift mutations occur in regions of repetitive DNA, we have designed a system to examine frameshifts that occur in very short repeats (< 4 nucleotides) and noniterated sequences. We have examined the patterns of frameshifts at these sites and how the mismatch repair pathway acts to suppress these mutations. Together, the experiments presented here provide further insight into the different mechanisms that suppress and/or influence rates of oxidative mutagenesis and describe a system in which we have begun to characterize how frameshift mutations are generated at very short repeats and non-repetitive DNA.

Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. Top1 cleaves one strand of DNA, covalently associates with the 3’ end of the nick to form a Top1-cleavage complex (Top1cc), passes the intact strand through the nick and finally re-ligates the broken strand. The chemotherapeutic drug, Camptothecin, intercalates at a Top1cc and prevents the crucial re-ligation reaction that is mediated by Top1, resulting in the conversion of a nick to a toxic double-strand break during DNA replication or the accumulation of Top1cc. This mechanism of action preferentially targets rapidly dividing tumor cells, but can also affect non-tumor cells when patients undergo treatment. Additionally, Top1 is found to be elevated in numerous tumor tissues making it an attractive target for anticancer therapies. We investigated the effects of Top1 on genome stability, effects of persistent Top1-cleavage complexes and elevated Top1 levels, in Saccharomyces cerevisiae. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in reciprocal crossovers, three- to fifteen fold increase in mutagenesis and greatly increased instability within the rDNA and CUP1 tandem arrays. Increased Top1 levels resulted in a fifteen- to twenty-two fold increase in mutagenesis and increased instability in rDNA locus. These results have important implications for understanding the effects of CPT and elevated Top1 levels as a chemotherapeutic agent.