Homologous recombination is involved in transcription-coupled repair of UV damage in Saccharomyces cerevisiae

Abstract

To efficiently protect the integrity of genetic information, transcription is connected to nucleotide excision repair (NER), which allows preferential repair of the transcribed DNA strands (TS). As yet, the molecular basis of this connection remains elusive in eukaryotic cells. Here we show that, in haploids, the RAD26 gene is essential for the preferential repair of the TS during G1. However, in G2/M phase there is an additional RAD51-dependent process that enhances repair of TS. Importantly, the simultaneous deletion of both RAD26 and RAD51 led to complete abolishment of strand-specific repair during G2/M, indicating that these genes act through two independent but complementary subpathways. In diploids, however, RAD51 is involved in repair of the TS even in G1 phase, which unveils the implication of homologous recombination in the preferential repair of the TS. Importantly, the abolishment of NER, by abrogation of RAD1 or RAD14, completely stopped repair of UV damage even during G2/M phase. These results show the existence of functional cross-talk between transcription, homologous recombination and NER.

Figure 1

Strand bias for NER is more pronounced in G2/M phase of haploid cells. S. cerevisiae cells synchronized in G1 or G2/M were UV-irradiated and allowed to repair for the indicated periods of time, and then NER was analyzed by primer extension. (A) FACScan analysis showing the synchronization procedure. (B, C) Autoradiograms showing repair of the PDs formed along the GAL10 TS and NTS, respectively. Asterisks, nonspecific Taq polymerase arrests; sequencing reactions (T, C, G, A). The pyrimidine tracks on the left represent the main PD clusters, with the accompanying number in parentheses referring to the 5′ nucleotide of the cluster. (D–F) Quantitative analysis of PD removal from the GAL10 and ACT1 genes, respectively. Illustrated are the fractions (%) of PD removed at each repair time. Each data point corresponds to an average value for the repair of several PD clusters. Inter-lane loading differences were corrected as described in Materials and methods. Each error bar represents the standard deviation of at least three experiments.

Figure 2

Cell cycle-dependent TCNER is transcription-related. Cells were grown in glucose, synchronized either in G1 or in G2/M and then reincubated for dark repair. PD removal was assessed on both strands of the repressed GAL10 gene. (A) Autoradiograms, the legends are as in Figure 1. (B) Quantitative analysis, performed as described in Figure 1.

Figure 3

RAD26 inactivation abolishes strand-specific repair and sensitizes cells in G1. rad26Δ cells were synchronized in G1 or G2/M, UV-challenged and then reincubated for repair. (A) Autoradiograms, the legends are as in Figure 1. (B) Quantitative analysis, performed as described in Figure 1D. Dashed lines represent quantification results from Figure 1D (G2/M) that were included for comparison. (C) Survival curves. Synchronized WT and rad26Δ cells were UV-irradiated with increasing UV fluences, and the percentage of surviving cells corresponding to each UV fluence was determined. Each error bar represents the standard deviation of three different experiments.

Figure 4

RAD51 and RAD54, but not RAD50, are required for the preferential repair of the TS in the haploid G2/M cells. WT, rad50Δ, rad51Δ, and rad54Δ cells were synchronized in G1 or G2/M, UV-challenged with 200 J m⁻² (B, C) or 100 J m⁻² (D) and then reincubated for repair. (A) FACScan analysis showing G2/M synchronized rad51Δ cells. (B) Autoradiograms, the legends are as in Figure 1. (C, D) Quantitative analysis, performed as described in Figure 1D. Each error bar represents the standard deviation of three experiments.

Figure 5

Simultaneous deletion of RAD51 and RAD26 abolishes strand-specific repair in G2/M. WT as well as the double mutant rad26Δrad51Δ cells were synchronized in G2/M, UV-challenged (200 J m⁻²) and then reincubated for repair. (A) Autoradiograms, the legends are as in Figure 1. (B) Quantitative analysis, performed as described in Figure 1D. Dashed lines represent quantification results from Figure 4C (rad51, G2/M) that were included for comparison. (C) Site-specific removal of PDs from the indicated PD clusters of the GAL10 TS, after 2 h of dark repair: rad51Δ (dashed bars), rad26Δ (solid bars), rad26Δrad51Δ (open bars). Each error bar represents the standard deviation of three experiments.

Figure 6

RAD51 is required for the preferential repair of the TS in diploid cells. WT and rad51Δ diploid cells were UV-challenged (200 J m⁻²) and then reincubated for repair. (A) Log phase cells. Illustrated is quantitative analysis of site-specific removal of PD from the indicated PD clusters of the GAL10 TS, after 2 h of dark repair: WT (dashed bars), rad51Δ (solid bars). (B) G1 cells, DNA profiles by FACScan analysis. (C) G1 cells, Autoradiograms, the legends are as in Figure 1. (D) G1 cells. Quantitative analysis, performed as described in Figure 1D. Each error bar represents the standard deviation of three experiments.

Figure 7

RAD14 deletion abolishes repair of the TS in G2/M phase. rad14Δ cells were synchronized in G2/M, irradiated with a UV fluence of 200 J m⁻², and then reincubated for dark repair. The legends of the autoradiogram are as in Figure 1.