Authors:	Villagomez Torres, Maria Beatriz
Title:	High-resolution analysis of replication-associated lesions bypass in time and space
Online publication date:	23-Feb-2023
Year of first publication:	2023
Language:	english
Abstract:	To maintain genome stability, DNA must be replicated accurately during every cell cycle. Fork progression and DNA synthesis depend on the coordinated action of the replisome. Nonetheless, replisomes frequently encounter various obstacles and, if not properly resolved, these can slow down or stall the progression of the replication fork. Even though the mechanisms of how the replisome responds to lesions are not completely understood, a few in vitro studies have implied mechanistic differences between the synthesis of lagging and leading DNA strands. The current challenge is to understand how the replisome responds to damage in vivo; therefore, the aim of this thesis was to comprehend the cellular responses to site-specific lesions found in lagging and leading strands in Saccharomyces cerevisiae. Additionally, to gain further insights into damage bypass, post-replication repair was investigated to understand its temporal and spatial organisation across the genome. To gain insights into how leading and lagging strands are replicated in the presence of polymerase-stalling lesions, the distribution of labour of the main DNA polymerases was investigated and the synthesis of daughter strands analysed under damaging conditions. Utilising a system that inflicts abasic sites at a single locus on chromosome VI in budding yeast while monitoring DNA polymerase usage, first, evidence for the role of Polymerase δ in the synthesis of damaged leading strands was provided. Secondly, by combining Next-Generation Sequencing approaches, I showed that both leading and lagging strands remained mostly un-replicated in the presence of damage. Moreover, it was observed that replication re-started downstream of the lesion, possibly indicating that de-novo re-priming events occurred in both strands. Additionally, the presence of abasic sites altered the loading of core replisome proteins at the damaged site. Finally, the experimental approach used during this study generated high genomic instability, which made it difficult to conclude on the dynamics of the replisome under DNA damaging conditions. To define the distribution of post-replication repair and to understand how cells maintain balance between translesion synthesis and template switching, a doxycycline repression system for depletion of Rad18 was applied. This confirmed that template switching is the preferred pathway over translesion synthesis in bypassing lesions induced by the alkylating agent methyl methanesulfonate. Interestingly, the genome-wide analysis of post-replication tracts indicated biases of template switching and translesion synthesis mostly towards regions within early firing origins as well as a greater contribution on leading strands than on lagging strands.
DDC:	000 Allgemeines 000 Generalities 570 Biowissenschaften 570 Life sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 10 Biologie
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-8787
URN:	urn:nbn:de:hebis:77-openscience-9c651b7d-7aaa-45c9-ab9e-8afb32775c198
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	http://rightsstatements.org/vocab/InC/1.0/
Extent:	xx, 158 Seiten ; Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen