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Title: Effect of naturally occurring DNA modifications on DNA structure and packaging
Author: Li, Zhe
ISNI:       0000 0004 7660 883X
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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In eukaryotes, the genomic double-stranded DNA (dsDNA) coils around histones to form nucleosomes. Arrays of these nucleosomes bundle together to generate chromatin. Most DNA-related processes require interactions between chromatin-protected DNA and cellular machinery. Access of cell machinery to genomic DNA is partially regulated by the position and stability of nucleosomes, which may be influenced by changes in nucleosomal DNA. DNA is composed of adenine (A), guanine (G), cytosine (C), thymine (T) nucleotides and their derivatives. It has been shown that some C derivatives participate in directing multiple biological processes, and aberrant modification patterns are often linked to diseases. It has been proposed that T derivatives exhibit similar effects. This thesis focuses on elucidating the effect of naturally occurring DNA modifications on the properties of dsDNA and nucleosomes. dsDNA sequences systematically modified with various T derivatives were characterized using classical biophysical techniques to assess the effect of these DNA modifications. The results indicate that in the sequence context studied, 5-hydroxymethyluracil modifications destabilize dsDNA, while dense symmetrical 5-formyluracil (fU) modifications alter the dsDNA structure. These effects may provide clues to the differential protein recruitment observed in previous research. In vitro studies on nucleosome occupancy and stability revealed that 5-formylcytosine (fC) modifications have positive effects on nucleosome formation and stability compared to the unmodified counterpart by influencing the intrinsic biochemical and biophysical properties of the nucleosomes. These results provide casual links for the observation in vivo between fC and the increased nucleosome occupancy and positioning. In order to further understand the positional effect of fC on the nucleosomes, a method was developed for quick and reliable incorporation of C derivatives into dsDNA at desired positions. The positive effect of fC modifications on nucleosome occupancy and stability observed here has necessitated further studies to gain deeper insights into the biological functions of fC in the nucleosome context. Cryo-EM can be used to elucidate the structural foundation for the changes fC posts to nucleosome, and protein interacting assays will identify the cellular machineries specifically recruited/repulsed by fC-modified nucleosomes. The effect of DNA modifications elucidated by the above studies advances our understanding on the role that DNA modifications play in regulating cellular processes.
Supervisor: Balasubramanian, Shankar Sponsor: Wellcome Trust ; Cancer Research UK ; A*STAR (Singapore)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: nucleosome ; DNA Modifications ; DNA Structure ; DNA Packaging