Browsing by Subject "Genomic instability"

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Open Access
Large-scale Effectors of Gene Expression and New Models of Cell Division in the Haloarchaea.
(2015) Dulmage, Keely
Like most Archaea, the hypersaline-adapted organism Halobacterium salinarum exhibits characteristics from all three domains of life, including a eukaryotic histone protein, a universal propensity to genetic rearrangements, and homologs of bacterial cell division proteins. Here we investigate the ancestral function of histone protein in the Archaea. Transcriptomics, proteomics, and phenotypic assays of histone mutants determine that histone regulates gene expression and cell shape but not genome compaction in H. salinarum. We further explore the regulation of gene expression on a genome-wide scale through the study of genomic instability. Genomic deletions and duplications are detected through the meta-analysis of 1154 previously published gene expression arrays and 48 chromatin immunoprecipitation arrays. We discover that a 90 kb duplication event in the megaplasmid pNRC100 directly leads to increased gene expression, and find evidence that the chromosome is far more unstable than previously assumed. These events are all linked with the presence of mobile insertion elements. Finally, in response to questions generated by these experiments, we develop a novel time-lapse protocol for H. salinarum and ask basic questions about single cell dynamics during division. Fluorescent labeling of homologs to bacterial cell division proteins confirms their involvement in cell division but localization dynamics contradict the basic bacterial model. The discovery of unusual facets of morphology during cell division is consistent with these novel protein dynamics and opens up new avenues of inquiry into archaeal cell division.
Open Access
Very high levels of misincorporated ribonucleotides increase Topoisomerase1 related genome alterations
(2017-05-09) Zhang, Lijia
A combination of high-resolution mapping of genomic rearrangements throughout the genome using microarrays, and measurements of loss of heterozygosity (LOH) on the right arm of chromosome IV were used to examine the effects of misincorporated ribonucleotides (rNMPs) on genome stability in diploid Saccharomyces cerevisiae strains. The effects of three types of mutations were examined in my analysis. Strains with a top1 mutation lack the Topoisomerase 1 enzyme, an enzyme that is involved in relaxing supercoils and in the removal of rNMPs from the genome. Strains with the rnh201 mutation lack RNase H2, an enzyme that removes both R-loops (RNA-DNA hybrids formed during transcription) and misincorporated rNMPs. Lastly, strains with the pol2-M644G mutation have a mutant form of DNA polymerase ε that misincorporates about 10-fold more rNMPs than the wild-type enzyme. My analysis of genetic instability in single mutants and various combinations of double and triple mutants shows that high levels of misincorporated rNMPs elevate mitotic recombination. Since mitotic recombination events are initiated in yeast by double-stranded DNA breaks (DSBs), my results suggest that high levels of misincorporated rNMPs result in elevated levels of DNA breaks.