HDF1 and RAD17 genes are involved in DNA double-strand break repair in stationary phase Saccharomyces cerevisiae

Abstract

DNA repair, checkpoint pathways and protection mechanisms against different types of perturbations are critical factors for the prevention of genomic instability. The aim of the present work was to analyze the roles of RAD17 and HDF1 gene products during the late stationary phase, in haploid and diploid yeast cells upon gamma irradiation. The checkpoint protein, Rad17, is a component of a PCNA-like complex-the Rad17/Mec3/Ddc1 clamp-acting as a damage sensor; this protein is also involved in double-strand break (DBS) repair in cycling cells. The HDF1 gene product is a key component of the non-homologous end-joining pathway (NHEJ). Diploid and haploid rad17Delta/rad17Delta, and hdf1Delta Saccharomyces cerevisiae mutant strains and corresponding isogenic wild types were used in the present study. Yeast cells were grown in standard liquid nutrient medium, and maintained at 30 degrees C for 21 days in the stationary phase, without added nutrients. Cell samples were irradiated with (60)Co gamma rays at 5 Gy/s, 50 Gy <or= Dabs <or= 200 Gy. Thereafter, cells were incubated in PBS (liquid holding: LH, 0 <or= t <or= 24 h). DNA chromosomal analysis (by pulsed-field electrophoresis), and surviving fractions were determined as a function of absorbed doses, either immediately after irradiation or after LH. Our results demonstrated that the proteins Rad17, as well as Hdf1, play essential roles in DBS repair and survival after gamma irradiation in the late stationary phase and upon nutrient stress (LH after irradiation). In haploid cells, the main pathway is NHEJ. In the diploid state, the induction of LH recovery requires the function of Rad17. Results are compatible with the action of a network of DBS repair pathways expressed upon different ploidies, and different magnitudes of DNA damage.

Fig. 1

Bar diagrams showing the relative frequency of survival for different absorbed doses. Samples were plated in nutrient medium either immediately after irradiation (black bars: t = 0 h) or after 24 h incubation in buffer (white bars: t = 24 h). a: WS9154 wild type; b: rad17/rad17 mutant strain. Error bars correspond to binomial confidence intervals (p ≤ 0.05)

Fig. 2

Left panel laser densitograms corresponding to diploid WS9154 strain. (1) control sample t = 0 h; (2) irradiated sample, D = 100 Gy, t = 0 h; (3) irradiated sample, D = 100 Gy, t = 24 h. The right panel shows the corresponding gel photograph after electrophoretic chromosomal separation. An increase of DNA fluorescence between bands indicates DNA fragmentation (see Section 2)

Fig. 3

Left panel laser densitograms corresponding to the rad17/rad17 diploid mutant strain. (1) control sample t = 0 h; (2) irradiated sample, D = 100 Gy, t = 0 h; (3) irradiated sample, D = 100 Gy, t = 24 h. The right panel shows the corresponding gel photograph after electrophoretic separation. An increase of DNA fluorescence between bands indicates a higher DNA fragmentation than that observed in wild type (Fig. 2)

Fig. 4

Bar diagrams showing the relative frequency of survival for different absorbed doses. Samples were plated in nutrient medium either immediately after irradiation (black bars: t = 0 h) or after 24 h incubation in buffer (white bars: t = 24 h). a: SX46 haploid wild type; b: SX46 hdf1Δ mutant strain. Error bars correspond to binomial confidence intervals (p ≤ 0.05)

Fig. 5

Left panel laser densitograms corresponding to the haploid SX46 wild type strain. (1) control sample t = 0 h; (2) irradiated sample, D = 100 Gy, t = 0 h; (3) irradiated sample, D = 100 Gy, t = 24 h. The right panel shows the corresponding gel photograph after electrophoretic separation. An increase of DNA fluorescence between bands indicates DNA fragmentation

Fig. 6

Left panel laser densitograms corresponding to SX46 hdf1Δ haploid mutant strain. (1) control sample t = 0 h; (2) irradiated sample, D = 100 Gy, t = 0 h; (3) irradiated sample, D = 100 Gy, t = 24 h. The right panel shows the corresponding gel photograph after electrophoretic separation. An increase of DNA fluorescence between bands indicates a higher DNA fragmentation than that observed in wild type (Fig. 5)

Fig. 7

Rad17 and Hdf1 play essential roles in maintaining genome stability and chromosomal recovery in late stationary phase cells after exposure to gamma rays. Damage sensors are MRX (top, left) and Rad17-complex (top, right). In the haploid state the most probable DSBs repair pathway is NHEJ (MRX-Hdf1-Hdf2-LigIV-Lif4). In the diploid state, homologous recombination pathways depend on RAD17-MEC1-RAD53-RAD55-RAD51 gene products, as well as on MRX-Rad52 securing LH survival, and DSB repair [7, 10, 19]. Mec1/hATR and Tel1/hATM kinases are at the core of the network and interact with sensors at DNA damage and with downstream effectors, enabling the signal amplification [3, 17, 18, 21, 22]. (LigIV ligase IV, Lif4 LigIV co-factor)