Assessing the Efficiency of Double-Strand Break Repair Mediated by Homologous Recombination and Non-homologous End-Joining Pathways in Saccharomyces cerevisiae

Abstract

The DNA double-strand breaks (DSBs) generated by exogenous and endogenous factors are repaired by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). These two pathways compete for DSB repair, and the choice of pathway depends on the context of the DNA lesion, the stage of the cell cycle, and the ploidy in the yeast Saccharomyces cerevisiae. However, the mechanistic details of the DSB repair pathway choice and its consequences for S. cerevisiae genome stability remain unclear. Here, we present PCR-based and cell-based assays as well as data analysis methods to quantitatively measure the efficiency of HR and NHEJ at DSBs in S. cerevisiae. An intermolecular recombination assay between plasmid and chromosomal DNA involving G-quadruplex DNA and a "suicide-deletion" assay have been utilized to evaluate the efficiency of HR and NHEJ, respectively. These streamlined protocols and optimized growth conditions can be used to identify the NHEJ- and HR-deficient S. cerevisiae mutant strains. Key features • Optimized protocol for intermolecular recombination involving G-quadruplex-forming DNA sequences derived from recombination hotspots in S. cerevisiae. • Optimized protocol to quantify the efficiency of NHEJ in S. cerevisiae. • Quantitative assessment of HR and NHEJ efficiency and data validation. This protocol is used in: eLife (2024), DOI: 10.7554/eLife.96933.3.

Keywords: Double-strand break; G-rich sequence (G4 DNA); Homologous recombination; Non-homologous end joining.

Figure 1.. Plasmid-chromosome recombination assay to assess G4 DNA-induced genome instability.

(A) Schematic representation of plasmid-chromosome recombination in W1588-4C and its derivatives. (B) Representative plate images of URA3-positive S. cerevisiae colonies (as papillae) obtained for the respective strains on YNB agar medium lacking uracil. (C) Graph representing the rate of recombination observed for different strains. Statistical analysis was performed in GraphPad Prism (v. 5.0) using one-way ANOVA and Tukey’s post-hoc tests. ns, not significant; ****p < 0.0001 compared with wild-type (WT) control.

Figure 2.. Assessment of non-homologous end-joining (NHEJ) using the “suicide-deletion” assay.

(A) Schematic representation of the experimental workflow (drawn in BioRender). (B) FALCOR software webpage indicating input requirements. ‘r’ = number of colonies/mL observed on reporter plates; ‘N’ = number of colonies/mL observed on reference plates. (C) Quantification of the rate of end-joining in WT, rev7Δ, and ku70Δ strains. Data is representative of three independent experiments. Statistical analysis was performed using one-way ANOVA and Dunnett’s multiple comparisons test, where ***p < 0.001, ****p < 0.0001 compared with wild-type (WT) control. Error bars represent mean ± SEM.

Figure 3.. Validation of plasmid-chromosome recombination assay.

(A) Schematic representation of plasmid-chromosome recombination in the S. cerevisiae strain YPH499 and its derivatives. (B) Representative plate images of URA3-positive S. cerevisiae colonies of the yeast strain YPH499 carrying ura3-52 and pif1-m2 single or double mutations. The ura3-52 strains were transformed with either an empty vector (ura3-52/pFAT10) or the pFAT10-G4 vector. Similarly, ura3-52 pif1-m2 strains were transformed with pFAT10-G4 vector. (C) Graph represents the rate of HR observed in indicated strains. Statistical analysis was performed in GraphPad Prism (v. 5.0) using one-way ANOVA and Tukey's post-hoc tests, where *p < 0.05, as compared with the wild-type (WT) control.