Abstract
In order to explore the mechanisms employed by living cells to deal with DNA alterations, we have developed a method by which we insert a modified DNA into a specific site of the yeast genome. This is achieved by the site-specific integration of a modified plasmid at a chosen locus of the genome of Saccharomyces cerevisiae, through the use of the Cre/lox recombination system. In the present work, we have used our method to insert a single UV lesion into the yeast genome, and studied how the balance between error-free and error-prone lesion bypass is regulated. We show that the inhibition of homologous recombination, either directly (by the inactivation of Rad51 recombinase) or through its control by preventing the polyubiquitination of PCNA (ubc13 mutant), leads to a strong increase in the use of Trans Lesion Synthesis (TLS). Such regulatory aspects of DNA damage tolerance could not have been observed with previous strategies using plasmid or randomly distributed DNA lesions, which shows the advantage of our new method. The very robust and precise integration of any modified DNA at any chosen locus of the yeast genome that we describe here is a powerful tool that will enable the exploration of many biological processes related to replication and repair of modified DNA.
Highlights
Various exogenous and endogenous agents pose a constant threat to the genome of all organisms, resulting in DNA modifications such as abasic sites, DNA adducts [1], DNA crosslinks, DNA–protein crosslinks [2], presence of ribonucleotides [3]
Recombination between LE and RE lox mutants produces a wild-type loxP site as well as a LE+RE double mutant lox site that is not recognized by Cre [25], preventing excision of the plasmid once integrated into the chromosome
Since our system was designed to explore the balance between error-prone and error-free lesion bypass pathways, we investigated the role of PCNA ubiquitination on the bypass of our UV lesions
Summary
Various exogenous and endogenous agents pose a constant threat to the genome of all organisms, resulting in DNA modifications such as abasic sites, DNA adducts [1], DNA crosslinks (intra- or inter-strand), DNA–protein crosslinks [2], presence of ribonucleotides [3] Unrepaired, these modifications present a serious challenge to a cell, as they may impair replication or give rise to deleterious mutations. To complete replication and maintain cell survival in the presence of residual DNA damages, cells have evolved two DNA Damage Tolerance (DDT) mechanisms: (i) Translesion Synthesis (TLS), employing specialized DNA polymerases able to insert a nucleotide directly opposite the lesion This pathway is potentially mutagenic due to the miscoding nature of most damaged nucleotides and to the low fidelity of the TLS polymerases (reviewed in [5]); (ii) Damage Avoidance (DA, named strand switch, copy choice or homology directed gap repair), a generally error-free pathway where the cells use the information of the sister chromatid to circumvent the lesion in an error-free manner (reviewed in [6]). The balance between error-free and error-prone mechanisms is important since it defines the level of mutagenesis during lesion bypass
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