iDamage: a method to integrate modified DNA into the yeast genome

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 yeast genome, 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 poly-ubiquitination of PCNA (ubc13 mutant), leads to a strong increase in the use of TLS. Such regulatory aspects of the DNA damage tolerance could not have been observed with previous strategies using plasmid or randomly distributed 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 allow exploration of many biological processes related to replication and repair of modified DNA.