Author : MaryElizabeth Stein
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (142 download)
Book Synopsis G-Quadruplexes and Inflammation by : MaryElizabeth Stein
Download or read book G-Quadruplexes and Inflammation written by MaryElizabeth Stein and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Accurate and efficient genome duplication is necessary to preserve genome stability. Two factors that challenge the genome are repetitive sequences that form secondary DNA structures (non-B DNA) and chronic inflammatory features like oxidative stress. Defects in replication mechanisms and/or repair pathways to resolve these blocks can lead to stalled replication forks, increased mutagenesis, DNA breaks, and, ultimately, genome instability. Therefore, DNA polymerase recruitment, in coordination with DNA repair, is critical in the cellular response to non-B DNA and oxidative stress. Importantly, specialized polymerase recruitment is implicated in overcoming stalling at and synthesizing through non-B DNA and oxidative DNA lesions. My dissertation looks at contributions by these determinants to genome instability independently: (1) the impact to DNA polymerase fidelity by one type of non-B DNA, G-quadruplexes (G4s) and (2) gene expression changes of two specialized DNA polymerases and histological disease activity in chronically inflamed tissues. G4s are evolutionarily conserved and function in a wide variety of cellular processes. G4s can also compromise genome integrity and are sources of small- and large-scale mutagenesis. However, the precise impact of G4s on human DNA polymerase fidelity that contributes to this mutagenesis is not known. I used an in vitro forward mutation assay to investigate the fidelity of human replicative DNA polymerase delta (pol [delta]) and specialized polymerases kappa ([kappa]) and eta ([eta]) during synthesis of G4 motifs that differ in sequence, topology, and stability. I observed both small (e.g., frameshifts) and large (e.g., deletion of the G4 and part of the flanking sequence) polymerase errors within and surrounding the G4s. These errors were dependent on the polymerase, G4 topology and stability. Notably, the frequency of large-scale errors increased in substrates containing G4 motifs with parallel strands. Pol [eta] errors occurred in the 3' sequences flanking the stable, parallel G4 motifs, whereas pol [kappa] errors were frameshifts within the G-tracts of these stable G4 motifs. In silico analysis showed that most polymerase errors are predicted to maintain the G4 structure, and pol [kappa] may better maintain a stable, parallel G4 compared to pol [eta]. Both pol [eta] and [kappa] can also facilitate the bypass of oxidative DNA lesions. However, the roles of these polymerases in inflammatory diseases remain undefined. Wide-spread tissue damage due to oxidative stress, including alterations in DNA, proteins, and lipids, has been demonstrated in mouse models and patient colonic biopsies of inflammatory bowel disease (IBD). Using ulcerative colitis (UC), a form of IBD, as a chronic inflammatory disease model, I retrospectively assessed three groups of non-cancerous colon tissues: (1) UC + high grade dysplasia (HGD)/cancer (Progressors), (2) UC with no HGD/cancer (Nonprogressors), and (3) healthy Controls. In this discovery phase study, I measured pol [eta] gene (POLH) and pol [kappa] gene (POLK) expression in RNA isolated from frozen tissues of two to three colon regions per patient. Microscopic inflammation was evaluated by pathologists in formalin-fixed, paraffin embedded tissues from each patient using a histological index, the Geboes score. Importantly, I found context-dependent differential expression of POLH and POLK genes. POLH expression was upregulated in UC tissues compared to Controls, independent of inflammation severity, whereas POLK expression was downregulated in tissues with active inflammation. I determined distinct disease activity differences between Progressor and Nonprogressor colon tissues, where more Progressor tissues had chronically inactive disease. Differences in histological inflammation suggest that underlying molecular differences may distinguish between UC patient groups. Taken together, my work expands on our understanding of how G4s and chronic inflammation can challenge genome integrity. G4 motif heterogeneity differentially impacts DNA polymerase fidelity, creating mutational hotspots. Histological assessment of disease activity in UC colon tissues underscores important changes induced by microscopic inflammation. Importantly, further studies are needed to determine the mechanisms of pol [eta] and pol [kappa], which may function differently at sites of G4s and oxidative damage.
