Saccharomyces cerevisiae-based system for studying clustered DNA damages

Abstract

DNA-damaging agents can induce clustered lesions or multiply damaged sites (MDSs) on the same or opposing DNA strands. In the latter, attempts to repair MDS can generate closely opposed single-strand break intermediates that may convert non-lethal or mutagenic base damage into double-strand breaks (DSBs). We constructed a diploid S. cerevisiae yeast strain with a chromosomal context targeted by integrative DNA fragments carrying different damages to determine whether closely opposed base damages are converted to DSBs following the outcomes of the homologous recombination repair pathway. As a model of MDS, we studied clustered uracil DNA damages with a known location and a defined distance separating the lesions. The system we describe might well be extended to assessing the repair of MDSs with different compositions, and to most of the complex DNA lesions induced by physical and chemical agents.

Fig. 1

Tester strain building: diploid 27.6.1. a Partial map of the ADE2 locus and surrounding regions on chromosome XV. Coordinate positions relevant to the work presented here are shown. In the haploid strain BY4741, the LEU2 selectable marker (red segment) was inserted between pos. 565631 and 566372, generating the partial deletion allele ade2(Δ1-561). Similarly, in the haploid strain BY4742, an ade2(Δ773-1716) allele was generated by inserting the HIS3 selectable marker (green segment) between the pos. 564398 and 565419. Both these ade2 allelic forms are null (the numbers that follow the Δ symbol indicate the deleted residues in the ADE2 ORF). The position of the oligos A and H is noted since they were used to characterize, by PCR, the profile of the wild-type strain (Supplementary Fig. 1). b Schematic of the crossing of the ade2 mutant to generate the diploid strain 27.6.1. The two haploids, BY4741 ade2(Δ1-561), and BY4742 ade2(Δ773-1716) were crossed and the diploids formed were selected by complementation. Noticeably, the chromosomes of couple XV have a region of shared homology in the ADE2 locus, spanning coordinates pos. 565419 to 565631. Further, the position of the oligos A and H is no more the one reported for the wild-type strain, but is in accord with the insertion of the selectable markers, HIS3 and LEU2

Fig. 2

Linear cassette(s) building from vector pMM-25. The “warehouse” vector pMM-25 was SmaI-cut, and the 1.3-Kb cassette purified by agarose gel electrophoresis using Nucleospin ExtractII columns (Machery Nagel, Bethlehem, USA). A further double enzymatic digestion (BglII/BanI) of the 1.3-Kb cassette generated three products (37, 539, and 729 bp), the last two of which were purified and used in the ligase reactions for building the cassettes

Fig. 3

Transformation experiments with linear cassettes. a Schematic representation of the chromosomal context targeted by the linear cassette with the location of the oligos used to characterize the outcomes. The oligo mix A/H was used for PCR-profiling the transformants, and the mix I/L was used to amplify and clone the sequence spanning a relevant part of the insertion point. In the diploid tester strain, 27.6.1, the PCR with oligo mix A/H gives 2,380 and 3,750 bp bands, while the mix I/L yields a 669 bp product. b Summary of PCR reactions with oligo mix A/H on His⁻ transformants. Total reactions were loaded on 0.8% agarose gels and the run (5 V/cm, 3 h) was followed by ethidium bromide staining. Samples were loaded as indicated above each lane. In lanes L and T were loaded the molecular weight marker (1 Kb Promega, MD, USA) and the untransformed 27.6.1 strain, respectively. Left: all tested transformants from the experiment with control undamaged cassette (CCC) and single-strand damaged ones (SSD1 and SSD2) exhibit the parental profile of the untransformed tester strain 27.6.1 (2,380, 3,750 bp bands). Right: all tested transformants from the experiments with linear cassettes having cluster damages (Cluster +1 and Cluster −1) gave four non-parental profiles (indicated as α, β, γ, and δ) that are compatible with the predicted HR repair outcomes (Supplementary Fig. 4)