The Saccharomyces cerevisiae Rad6 postreplication repair and Siz1/Srs2 homologous recombination-inhibiting pathways process DNA damage that arises in asf1 mutants

Abstract

The Asf1 and Rad6 pathways have been implicated in a number of common processes such as suppression of gross chromosomal rearrangements (GCRs), DNA repair, modification of chromatin, and proper checkpoint functions. We examined the relationship between Asf1 and different gene products implicated in postreplication repair (PRR) pathways in the suppression of GCRs, checkpoint function, sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS), and ubiquitination of proliferating cell nuclear antigen (PCNA). We found that defects in Rad6 PRR pathway and Siz1/Srs2 homologous recombination suppression (HRS) pathway genes suppressed the increased GCR rates seen in asf1 mutants, which was independent of translesion bypass polymerases but showed an increased dependency on Dun1. Combining an asf1 deletion with different PRR mutations resulted in a synergistic increase in sensitivity to chronic HU and MMS treatment; however, these double mutants were not checkpoint defective, since they were capable of recovering from acute treatment with HU. Interestingly, we found that Asf1 and Rad6 cooperate in ubiquitination of PCNA, indicating that Rad6 and Asf1 function in parallel pathways that ubiquitinate PCNA. Our results show that ASF1 probably contributes to the maintenance of genome stability through multiple mechanisms, some of which involve the PRR and HRS pathways.

FIG. 1.

HU and MMS sensitivities of mutants lacking Asf1 and PRR pathway components. (A) Sensitivity of mutants to chronic exposure to HU. Serial dilutions of cells were plated onto YPD medium containing HU as indicated and incubated at 30°C for 2 to 3 days. (B) Sensitivity of mutants to chronic exposure to MMS. Conditions were the same as for panel A except that the plates contained the indicated concentrations of MMS instead of HU. (C) Mutants lacking Asf1 and PRR components are not sensitive to killing by acute exposure to HU. Cells were exposed to 200 mM HU in culture for 2 h, washed, and plated on YPD. The data are expressed as the percentage of viable cells (CFU) that survive acute HU treatment relative to the viable cells present in the culture at the time of addition of HU. The small increase in the number of CFU after treatment reflects the fact that there is some cell division that occurs during the early phase of growth after the addition of HU. Error bars indicate standard deviations.

FIG. 2.

S-phase progression of asf1, PRR, and Siz1 pathway mutants. The rates of S-phase progression of the indicated mutants after release from α-factor arrest were monitored using FACS analysis. The proportion of cells in G₂/M was determined at the 40-min time point, at which at least 80% of wild-type cells have reached G₂/M. The values presented are the averages of those from at least three experiments, and the error bars indicate the standard deviations.

FIG. 3.

Spontaneous checkpoint activation in mutants lacking Asf1 and PRR pathway components. Checkpoint activation was monitored in live cells by counting the number of small budded S-phase cells with Ddc2.GFP foci using confocal microscopy. The experimental results are quantified for single and double mutants and are expressed as the percentage of cells containing Ddc2.GFP foci. The values presented are the averages of those from at least three experiments, and the error bars indicate the standard deviations.

FIG. 4.

Modification of PCNA in untreated cultures depends mostly on Rad6 and partially on Asf1. α-Factor-synchronized cultures of the indicated mutants were released into YPD for 40 min, and modification of PCNA was analyzed by Western blotting to detect the protein A tag. Upper panel, short exposure; lower panel, overexposed Western blot.

FIG. 5.

Modification of PCNA in MMS-treated cells depends on both Rad6 and Asf1 and the K164 residue of PCNA. (A) α-Factor-synchronized cultures of the indicated mutants were released into YPD supplemented with 0.1% MMS, and samples were taken at the indicated time points. Cells were lysed, and modification of PCNA was analyzed by Western blotting to detect the protein A tag. (B) α-Factor-synchronized cultures of the indicated strains containing the TAF-tagged pol30-119 allele were released into YPD supplemented with 0.1% MMS, and samples were taken at the indicated time points. Cells were lysed, and modification of PCNA was analyzed by Western blotting to detect the protein A tag.

FIG. 6.

Ubiquitination of PCNA in MMS-treated cells depends on both Rad6 and Asf1. Wild-type cells, rad6 and asf1 single mutants, and asf1 rad6 double mutants were grown to log phase and arrested in G₁ phase using α-factor. Subsequently, cultures were released into YPD containing the indicated amounts of MMS for 2 h. Equal numbers of cells were then lysed, and Pol30 was immunoprecipitated using FLAG antibodies to pull down His-FLAG-tagged Pol30 and analyzed by Western blotting using anti-SUMO, antiubiquitin, and anti-FLAG antibodies.

FIG. 7.

Model for the involvement of Asf1 in PCNA modification. (A) When the replication fork encounters a lesion in the DNA, the Rad6-Rad18 pathway is responsible for bulk monoubiquitination of PCNA, obscuring the involvement of Asf1. Monoubiquitin can serve as a seed for further ubiquitination by the Rad5-Mms2-Ubc13 pathway. Sumoylation of PCNA also occurs but is not dependent on Rad6. (B) In the absence of Rad6, some ubiquitination of PCNA still takes place, and this is dependent on Asf1 and an unidentified E2/E3 pair. The sensitivity of our assays is not high enough to determine whether polyubiquitination can still take place in the absence of Rad6 (dashed arrow), but sumoylation of PCNA can still occur and is independent of either Rad6 or Asf1. (C) In the absence of Siz1, sumoylation of PCNA does not occur, and the antirecombination function of Srs2 is not activated. (D) In the absence of Rad5-Ubc13, ubiquitination of PCNA mediated by Rad6-Rad18 still takes place, but polyubiquitination does not appear to occur (dashed arrow). Under the circumstances described for panels B, C, and D, HR is stimulated, resulting in the suppression of asf1-induced GCRs, which also involves a Dun1-dependent checkpoint.